Test systems and the use thereof for identifying and characterizing compounds

ABSTRACT

The invention relates to test systems which are based on transmembrane receptors from helminths and arthropods, and to the use thereof for identifying and characterizing substances which act on helminths, arthropods or which act on the calcium balance of organisms and/or cells. The invention furthermore relates to the use of a specific ligand in this test system and to the use thereof as anthelmintic or arthropodicidal active substance.

The present invention relates to test systems which are based ontransmembrane receptors from helminths and arthropods, and to the usethereof for identifying and characterizing substances which act onhelminths, arthropods or which act on the calcium balance of organismshost cells. The invention furthermore relates to the use of a specificligand in this test system and to the use thereof as anthelmintic orarthropodicidal active substance.

Parasitic helminths and arthropods represent a considerable healthproblem for humans and animals. In the agricultural sector alone, thecost for controlling or preventing the damage caused by such parasitesaround the world amounts annually to more than 7 billion DM (1997figure). A large number of active substances from a number of classes ofactive substances are available for the treatment of the endoparasitosesmainly caused by helminths, and of the ectoparasitoses primarily causedby arthropods. During the last two decades there has been an increase inthe importance in particular of active substances which simultaneouslyshow a very good action on a plurality of parasite species within aphylum (e.g. helminths), or even extend over various zoological phyla(e.g. helminths and insects). The former include inter aliabroad-spectrum anthelmintics such as, for example, the benzimidazoles orthe imidazothiazoles, while the macrocyclic lactones are included amongthe latter.

The last time a novel, broadly active group of active substances waslaunched on the market was about two decades ago, with the macrocycliclactones. However, a large number of endoparasite and ectoparasitespecies have now developed resistance to individual classes, and in somecases simultaneously to a variety of classes, of active substances.Hence there is a continuing and increasingly urgent need for thedevelopment of novel substances with antiparasitic activity.

The few promising novel groups of active substances being worked on atpresent include the cyclic depsipeptides which are the subject ofextensive patent (WO 93/19053, EP 626 376, WO 94/19334, WO 95/07272, EP626 375, EP 657 171, EP 657 172, EP 657 173) and research activities(Conder et al. 1995; Martin et al. 1996; Sasaki et al. 1992; Terada M1992; Samson-Himmelstjerna et al. 2000).

A number of studies to elucidate the mechanism of action of onerepresentative of this group of active substances, PF 1022A(cyclo(-D-Lac-L-MeLeu-D-PhLac-L-MeLeu-)₂) have already been described(cf. information on this in DE-A-197 04 024).

These included the identification and characterization of a specificbinding protein for cyclic depsipeptides and of the DNA sequence codingfor the protein from the sheep parasite Haemonchus contortus (DE-A-19704 024). Within the scope of the present invention, the functionalinteraction of the abovementioned protein, called HC110-R, inter aliawith the ligand BAY 44-4400(cyclo(-D-Lac-L-MeLeu-D-p-morpholinyl-PhLac-L-MeLeu-), as a furtherrepresentative of the cyclic depsipeptides, is described. For thispurpose, recombinant eukaryotic cell lines in which HC110-R, based onsequence ID No. 2 described in the earlier application DE-A-197 04 024,is expressed have been constructed.

The present invention is accordingly based in particular on the objectof providing, on the basis of transmembrane receptors of helminths andarthropods, preferably from nematodes and acarina, particularlypreferably from Trichostrongylidae, very particularly preferably fromHaemonchus spp., and especially on the basis of the receptor HC110-Rfrom H. contortus, test systems with a high throughput of test compounds(high throughput screening assays; HTS Assays).

Homologous proteins are regarded as being those proteins which have atleast 70% identity, preferably 80% identity, particularly preferably 90%identity, very particularly preferably 95% identity, with a sequence asshown in SEQ ID NO: 2 of the document DE-A-197 04 024, the contents ofwhich are to be expressly included in the present application, over alength of at least 20, preferably at least 25, particularly preferablyat least 30 consecutive amino acids and very particularly preferablyover the complete lengths thereof.

The degree of identity of the amino acid sequences is preferablydetermined with the aid of the GAP programme from the GCG programmepackage, Version 9.1, with standard settings (Devereux et al. 1984).

The object is achieved by providing polypeptides which exercise at leastone biological activity of a GPC receptor, and by providing a method forobtaining these polypeptides and by providing methods for identifyingcompounds with nematicidal and arthropodicidal activity.

Test systems based on recombinant microorganisms have already been usedmany times for identifying pharmaceutically active substances, interalia also with use of microorganisms which recombinantly expressparasitic genes (Klein and Geary 1997). However, to date, no systems inwhich parasitic transmembrane receptors are used as recombinantfunctional proteins in eukaryotic cells as targets have been disclosed.The test systems described in this invention can be used to identifythis novel class of receptors in high-throughput-screening (HTS) or inultra-HTS.

Recombinant expression of receptors from nematodes has ordinarily provedto be difficult. Thus, it has not to date generally been possible toexpress G-protein-coupled receptors (GPCR) from nematodes in such a waythat their functional properties (e.g. sensitivity to inhibitors)correspond to those of natural receptors.

Functional expression of receptors from helminths, in particularnematodes, and from arthropods in eukaryotic systems is of greatpractical significance, for example in the search for novelanthelmintics or arthropodicides.

The present invention is thus also based on the object of providing apossible way of expressing transmembrane receptors, especially GPCreceptors, from nematodes and arthropods and, based on this, to developa test system which makes it possible to identify novel substances withnematicidal and arthropodicidal activity.

The present invention thus relates in particular to the expression anduse of an orphan G-protein-coupled receptor from helminths andarthropods, preferably from nematodes and acarina, particularlypreferably from Trichostrongylidae, very particularly preferably fromHaemonchus and most preferably from the parasitic nematode H. contortus,as target protein for the efficient search for nematicidal activesubstances.

This receptor has been identified in a cDNA library which was obtainedfrom the gastrointestinal nematode H. contortus. The cDNA codes for aheptahelical transmembrane protein with a size of 110 kDa, which hasbeen referred to as HC110-R. The protein belongs to the secretin familyof G-protein-coupled receptors (GPCR) and shows great similarity withlatrophilin (FIG. 5).

The HC110-R Receptor as Target

The GPCR latrophilin was originally isolated from mammalian brain.Latrophilin has a molecular mass of 210 kDa and undergoespost-translational cleavage at residue 18 upstream of the firsttransmembrane segment, and thus consists of two non-covalently linkedsubunits. The p120 subunit contains the N-terminal hydrophilicextracellular portion, and the p85 subunit contains the seventransmembrane domains and the intracellular C-terminal region oflatrophilin, which is unusually large for GPCRs. Two close homologueshave recently been identified, latrophilin-2 and latrophilin-3 (see alsoFIG. 6). The latter is preferentially, like latrophilin-1 too, expressedin the brain, while latrophilin-2 is expressed ubiquitously with apreference for placenta, kidney, spleen, ovaries, heart and lung ofmammals (Ichtchenko et al. 1999; Sugita et al. 1998).

Although HC110-R is only half the size of the 210 kDa latrophilin, thesimilarity of the sequence also extends to a functional similarity. Theendogenous ligand for both receptors is still unknown, but bothlatrophilin and HC110-R are influenced by the artificial ligand α-LTX(alpha-latrotoxin).

If, for example, HEK-293 cells are transiently transfected withlatrophilin, the addition of α-LTX causes influx of external Ca²⁺, ascan be shown by means of an experimental design with radioactive ⁴⁵Ca²⁺.α-LTX also causes such a Ca²⁺ influx in HEK-293 cells which have beentransiently transfected with HC110-R, which can be observed for exampleby Ca²⁺ imaging (see also FIGS. 10 and 11).

This Ca²⁺ influx is, however, very complex. If the HC110-R is in theform of a construct with a C-terminal Green Fluorescent Protein (GFP)attachment, it is biphasic, i.e. it shows one change after about 3 andanother one after about 22 minutes. If, however, only an N-terminalMyc-His tag is put in front of HC110-R, the main influx is observed onlyabout 2-3 minutes after addition of α-LTX. The reason for this is notyet known, but the response to the α-LTX addition is specific, as shownby the subsequent statements:

-   1. The Ca²⁺ influx is not observable in untransfected cells and in    cells transiently transfected with a mouse β₂-adrenergenic receptor.-   2. The changes in [Ca²⁺]_(i) are dependent on the α-LTX dose (FIG.    10).-   3. The biphasic change requires influx of Ca²⁺, which takes place    through Ca²⁺ channels which can be blocked by Cd 2, especially those    of the L type, as is evident from their sensitivity to nifedipine.

The exact mechanism of the interaction of α-LTX with HC110-R and thetransduction of signals still requires explanation. It has been possibleto show for the example of latrophilin-1 that only a singletransmembrane domain is necessary for the Ca²⁺ influx caused by α-LTXinto HEK-293 cells (Kraspernov et al. 1999).

The fact that nifedipine blocks the effect of α-LTX in the systemdescribed here shows that the system is also suitable for identifyingnovel specific calcium channel blockers. Nifedipine belongs to a classof calcium channel agonists and antagonists which are classifiedaccording to their binding to a specific site of a calcium channel, e.g.on the basis of their binding to L-type channels. The principal threeclasses of calcium channel blockers (L type) are benzylacetonitriles(e.g. verapamil, WO 91/02497), benzothiazepinones (e.g. diltiazem) and1,4-dihydropyridine derivatives such as nifedipine, nivaldipine,nimodipine, nicardipine, isradipine, amlodipine, nitrendipine,felodipine or nisoldipine (Bacon et al. 1989; WO 90/09792). Anotherclass of L-type calcium channel blockers comprises 1,3-diphosphonates,e.g. belfosodil.

This invention therefore also relates to the use of α-LTX-bindingtransmembrane receptors for identifying novel calcium channel blockers.Heptahelical transmembrane receptors are particularly preferably usedfor this purpose, and they are particularly preferably G-protein-bindingreceptors. The use of transmembrane receptors of the secretin family isvery particularly preferred. Most preference is given to the use of theHC110-R receptor as shown in SEQ ID. NO: 2, and receptor proteins whichare 70%, preferably 80%, particularly preferably 90% and veryparticularly preferably 95% homologous thereto.

The available data also show that HC110-R is a target for the novelanthelmintic depsipeptide BAY44-4400. BAY44-4400 interferes with signaltransmission by α-LTX in HEK-293 cells which have been transfected withHC110-R (FIG. 12). This interference may derive from the ionophoricactivity of BAY 44-4400, as is typical of other depsipeptides, e.g.beauvericin, enniatin etc. (Geβner et al. 1996). However, BAY44-4400causes no changes in untransfected cells. In addition, BAY44-4400 doesnot interfere with the isoproterenol-induced signal transmission throughHEK-293 cells transfected with the mouse β₂-adrenergic receptor. Inaddition, BAY44-4400 acts as antagonist of α-LTX, while BAY44-4400 alonedoes not influence the Ca²⁺ concentration in HC110-R-transfected HEK-293cells.

The HC110-R Protein

The membrane protein described in DE 197 04 024 A1 was identified as apossible target protein for the anthelmintic active substance PF1022A.Possible target proteins were identified by preparing a γZAPII cDNAexpression library of the parasitic nematode Haemonchus contortus, andscreening the cDNA library using a conjugate of PF1022A and KLH (keyholelimpet hemocyanin), PF1022A-KLH, and polyclonal antibodies against thisconjugate (see Example 1). Examination of about 1.5×10⁶ non-amplifiedrecombinant clones revealed a cDNA clone with a length of 3036 bp whichhybridized with a 3.6 kb mRNA from H. contortus (see also Example 2).The RACE PCR technique was used to complete the 3′ and 5′ ends (Example4). Finally, a cDNA with a length of 3539 bp was obtained and wasreferred to as HC110-R. This cDNA codes for 986 amino acids (10 kDa),with the open reading frame starting with ATG¹⁰⁰ (FIG. 2). The startcodon is surrounded by a Kozak sequence for optimal initiation oftranslation. The derived molecular weight of the protein was confirmedby in vitro translation of HC110-R RNA transcribed in vitro.

The amino acid sequence of the HC110-R protein shows some particularfeatures. The extracellular N-terminal part of the protein consists of atotal of 535 residues. The N terminus comprises a signal peptide with alength of 21 amino acids and a cleavage site at position 18. This isfollowed by a lectin-like sequence (AA 22-125) and a so-called “Thrstretch” (AA 128-147) which is interrupted only by a serine in position144. Downstream of the “Thr stretch” there is a cysteine-rich motif withthe structure CX₉WX₁₂CX₉WXCX₅WX₉CX₃W (AA 166-221). In addition, theHC110-R protein contains seven hydrophobic α-helical transmembranedomains between residues 536 and 772. Directly upstream from thetransmembrane region there is a further 4-Cys region with the structureCXWWX₆WX₄CX₁₁CXC (AA 478-524). In the transmembrane region there arethree extracellular loops comprising the residues 587-597, 654-673 and743-749 and three intracellular loops (position 559-569, 627-636,696-724). The C terminus is 214 residues long (24 kDa) and contains aproline-rich part (AA 845-861) and a PEST region (AA 915-933). Finally,there are also three putative N-glycosylation sites at residues 26, 499and 862, and 14 putative phosphorylation sites in the derivedintracellular domains (see also FIG. 6).

Analysis of databases revealed 48% identity and 76% similarity of theHC110-R proteins with an unknown transmembrane protein (1014 AA) whichwas derived from the genomic clone B0457 (GenBank™ Accession NumberZ54306) of the nematode Caenorhabditis elegans (see also FIG. 3).

The term “identity” as used herein describes the number of sequencepositions which are identical in a so-called alignment. It is stated asa percentage of the alignment length.

The term “similarity” as used herein describes the similarity ofsequences on the basis of a similarity metric, that is to say a measureof how similar for example a valine is to be assumed to be to athreonine or to a leucine.

The term “homology” as used herein means evolutionary relationship, thatis to say if two homologous proteins have developed from a commonprecursor sequence. The term does not necessarily have anything to dowith identity or similarity, apart from the fact that homologoussequences are usually more similar, or have more identical positions inan alignment than do non-homologous sequences.

Comparison of the two sequences shows features common to both proteins,for example the lectin-like sequence, the “Thr stretch”, the Cys motifsand the PEST sequence. The greatest identity is to be found in thetransmembrane region (62%), whereas the identity is found to be lesspronounced in the N-terminal (44%) and C-terminal (50%) region.

In addition, the HC110-R protein has 20-30% identity with heptahelicalG-protein coupled transmembrane receptors (GPCR), especially with thesecretin subfamily. Comparisons of seven transmembrane domains show ahigh degree of identity and similarity in relation to structure andsequence between various GPCRs of the secretin subfamily and HC110-R(FIG. 4).

Latrophilin, a member of the secretin subfamily, from mammals such ashumans (GenBank™ Accession No.: E1360690), cattle (e.g. G416021, G416053and G4185804), and rats (U78105 or U72487) show somewhat greateridentity (31%) with HC110-R than do other secretin receptors. Inparticular, the transmembrane regions show an identity of 45-48%.HC110-R and the rat latrophilin-1 GPCR (U78105) of 1466 amino acidsdisplay common features, e.g. the lectin domain, the cysteine-richregion and a conserved 4-Cys motif in front of the transmembrane region.The latter was recently proposed to be proteolysis site of latrophilinand other large secretin GPCRs. By contrast, the N terminus of HC110-Rdoes not contain the olfactomedin region and the Pro/Thr region oflatrophilin, while latrophilin in turn does not have the “Thr stretch”of HC110-R.

The present application therefore also relates to the use ofG-protein-coupled transmembrane receptors with seven transmembranedomains from helminths for identifying substances with anthelminticactivity. Preference is given according to the invention to the use oftransmembrane receptors which can be assigned to the secretin subfamily.

Cellular Localization

Transient transfection experiments were carried out with an HC110-R-GFPfusion protein in various mammalian cell lines, for example COS-7 cellsor HEK-293 cells (FIG. 7). Heterologous expression was chosen because noH. contortus cell lines have yet been established.

The green fluorescent protein (GFP) from the pacific jellyfish Aequoreavictoria can be used for localizing proteins in living cells (see FIG.8). GFP is used at the cellular level as an in vivo reporter in order toindicate the frequency of a transient or stable transfection and at thesubcellular level for localizing proteins. Wild-type GFP is a 27 kDmonomer composed of 238 amino acids which emits green light with amaximum of 509 nm after excitation with UV light (360-400 nm; max. at395 nm) or blue light (440-480 nm; max. at 475 nm), without requiringexogenous substrates or cofactors for this (Chalfie et al. 1994). GFPcan thus be detected in vivo directly by fluorescence microscopy, andits fluorescence characteristics are essentially unchanged even whenpart of a fusion protein. EGFP (enhanced GFP) is a genetic variant ofthe wild-type GFP and is employed for the transfection of mammaliancells (Yang et al. 1996). The excitation maximum of EGFP has beenshifted to only one peak at 490 nm by replacing Ser⁶⁵ by Thr. Thevectors pEGFP-1 (GenBank Accession No.: U55763) and pEGFP-N3 (GenBankAccession No.: U55762) [Clontech, Palo Alto, Calif., U.S.A.] expressEGFP under the control of the strong constitutive CMV promoter and canbe used to fuse other proteins respectively to the N and C terminus ofEGFP.

Stable cell lines have the advantage over transient expression thatevery cell permanently expresses the desired protein, and isolation ofthe protein is possible after localization. In order to detect theprotein by fluorescence microscopy or with the aid of a Western blot, anantibody against the desired protein is required, or it is appropriateto choose an expression vector which, for example, fuses a Myc or Histag C-terminally to the actual protein in the correct reading frame,against which there are then antibodies which can be purchased, in mostcases even monoclonal ones.

The present invention likewise relates to cells which make stableexpression of the HC110-R receptor, and proteins homologous thereto,possible.

Effect of Alpha-Latrotoxin on the HC110-R Protein

To further substantiate the similarity between HC110-R and latrophilin,and to examine the functionality of the recombinantly expressed HC110-R,HEK-293 cells were transiently and stably transfected with HC110-R-GFPfusion protein and stimulated with alpha-latrotoxin (a-LTR).

Alpha-latrotoxin is a presynaptic neurotoxin which can be isolated fromblack widow (Latrodectus mactans) venom. It is known for its toxicityfor the central nervous system of vertebrates, where it induces thedepolarization of neurons through increasing [Ca²⁺]_(i) and stimulatinguncontrolled exocytosis of neurotransmitters. Thus, it has also beendisclosed that the effect of α-latrotoxin is mediated at least in partby latrophilin. It is moreover assumed that the toxicity of α-latrotoxinderives from its ability to interact with receptors which are coupled toGTP-binding protein (GPCR). These receptors normally mediate the effectof endogenous hormones or neuropeptides (Holz and Habener 1998).

In the present invention, the so-called Ca²⁺ imaging technique was usedin order to test the response of transfected HEK-293 cells to α-LTX, bydetermining the change in the Ca²⁺ present in the cell [Ca²⁺]_(i) (seeExample 24 and FIGS. 9, 10 and 11).

α-LTX causes a biphasic increase in [Ca²⁺]_(i). At a concentration of 75nM, α-LTX induces initially a very small increase of only 5±0.2 nM 2minutes after α-LTX addition and a larger, delayed increase of about220+14.9 nM Ca²⁺ after 22 minutes. As the α-LTX concentration increases(7.5 nM-120 nM), the first increase becomes larger, while the secondincrease diminishes. At an α-LTX concentration of 120 nM, the firstincrease reaches values of about 135±13.6 nM Ca²⁺, while the secondincrease falls to a value of 50+7.1 nM Ca²⁺. The same profile on use of90 nM and 120 nM α-LTX indicates saturation.

Transfection of HEK-293 cells with an N-terminal GFP-tagged HC110-Rconstruct and stimulation of them with 75 nM α-LTX results in a slightlyreduced 2nd peak. Finally, an only slightly diminished response to α-LTXhas also been found when the HC110-R protein has been provided with anN-terminal GFP tag. It can therefore be assumed that N- or C-terminalattachment of a GFP tag has a negligible effect on α-LTX binding andsubsequent signal transduction by HC110-R.

Cells which had not been transfected or had been only transientlytransfected with GFP show no response to α-LTX. If HEK-293 cells aretransiently transfected with other G-protein-coupled receptors withC-terminal GFP tags, e.g. the mouse β₂-adrenergic receptor, or the humanmuscarinergic H1 acetylcholine receptor, the increase in [Ca²⁺]_(i)caused by α-LTX at a concentration of 75 nM is only small (of about40+10.4 nM after about 20 minutes) or zero.

The increase in [Ca²⁺]_(i) caused by α-LTX may lead both to influx ofextracellular Ca²⁺ and to efflux of intracellular Ca²⁺. If extracellularCa²⁺ is removed with 2 mM EGTA before or after the addition of α-LTX,there is only a small increase in the [Ca²⁺]_(i) of HEK-293 cells whichexpress the HC110-R-GFP fusion protein. It is evident from this thatα-LTX primarily causes influx of extracellular Ca²⁺. This Ca²⁺ influx isnot based on simple diffusion but takes place with the aid of Ca²⁺channels in the plasma membrane.

Most of the Ca²⁺ channels involved in the Ca²⁺ influx are those of the Ltype, because 15 μM nifedipine is sufficient to depress significantlythe α-LTX-induced increase in [Ca²⁺]_(i). Moreover, the first increaseis completely inhibited, and the second increase falls from 267±12.7 nMto 30±5.4 nM.

The stable or transient HEK-293 cell line expressing the HC110-Rreceptor with a C-terminal Myc/His tag also responds dose-dependently toα-LTX (FIG. 10). 7.5 nM α-LTX are still too little to generate anα-LTX-induced Ca²⁺ influx in this case too.

However, even 25 nM α-LTX are sufficient to generate an increase in[Ca²⁺]_(i) of 130±38.0 nM after only 2 min. On addition of 75 nM α-LTXthere is a Ca²⁺ influx of 296±91.5 nM, which increases greatly likewise2 min after α-LTX addition and returns to its original Ca²⁺ content onlyafter 27 min. This Ca²⁺ signal resembles the second delayed Ca²⁺ peak ofHC110-R-GFP-transfected HEK-293 cells at the same α-LTX concentration(75 nM) in the height of the signal and in its profile, but the responsetakes place—as also with higher concentrations (90 nM and 120 nM) ofHC110-R-GFP-transfected cells—immediately after α-LTX addition.

The invention therefore likewise relates to the use of α-LTX as agonistof transmembrane receptors of the sekretin family from nematodes. α-LTXis preferably used as agonist of the HC110-R receptor as shown in SEQ IDNO: 2, and of receptor proteins which are 70%, preferably 80%,particularly preferably 90% and very particularly preferably 95%homologous thereto.

The present invention likewise relates to the use of α-LTX as anematicide.

The present invention likewise relates to the use of α-LTX in a methodfor identifying compounds with nematicidal or arthropodicidal activity,the compounds possibly being active as agonists or antagonists oftransmembrane receptors.

The present invention likewise relates to the use of α-LTX in a methodfor identyfing compounds which block calcium channels.

Effect of BAY 44-4400 on the Action of α-LTX

PF1022A exerts its effect on nematodes at concentrations in the range100-800 ng/ml, depending on the particular species (Terada, 1992). Inorder to investigate in each case any possible interaction betweenPF1022A with HC110-R and the signal transmission mediated by HC110-R,BAY44-4400, which is a readily soluble derivative of PF1022A, was usedin the following Ca²⁺ imaging experiments in HEK-293 cells loaded withFURA-2 and transfected with HC110-R-GFP.

At a concentration of 400 ng/ml, neither the very effective nematicideBAY44-4400 nor the virtually ineffective antipode PF1022-001 induces aCa²⁺ response in HC110-R-GFP-transfected HEK 293 cells, even if thecells had been preincubated with the active substance for 90 minutes. Incontrast to this, both substances affect the signal transmissiondependent on α-LTX, although to differing extents (FIGS. 12 and 14). Inthe presence of 4 ng/ml BAY44-4400, α-LTX induces only a small Ca²⁺increase with a maximum of 44±6.0 nM Ca²⁺ after 14 minutes (FIG. 12). Inthe presence of PF1022-001, however, α-LTX causes a larger increase of103±11.5 nM Ca²⁺ after 6 minutes (FIG. 10B). In another approach, thecells were preincubated with either 4 ng/ml or 400 ng/ml BAY44-4400 andPF1022A for 90 minutes, and the active substances were then removedbefore the cells were stimulated with α-LTX. HEK-293 cells preincubatedwith PF1022A showed no significant change in their response to α-LTX.Only BAY44-4400 affected the sensitivity of the cells to α-LTX. At aBAY44-4400 concentration of 4 ng/ml, α-LTX induced a Ca²⁺ increase(95±20.5 nM Ca²⁺) 19 minutes before a stable Ca²⁺ level was reached. At400 ng/ml BAY44-4400, α-LTX caused an increase in the Ca concentrationto only about 65+7.5 nM Ca²⁺. In addition, this small increase wasshifted by 12 minutes.

In order to verify the specificity of these results, additionally theeffect of 1 mM carbachol on the endogenous, natural M1-R was measured inuntransfected HEK-293 cells. The presence of 400 ng/ml BAY44-4400 didnot change the response of the cells. Nor was the stimulation induced byisoproterenol and arecoline of the endogenous, natural β₂-R or of thenicotinic cholinergic receptor in HEK-293 cells affected by BAY44-4400in any way.

Finally, within the scope of the present invention, the effect of BAY44-4400, a more soluble variant of PF1022A, on the HC110-R protein inHEK-293 cells which transiently or stably express an HC110-R proteinprovided with C-terminal GFP was investigated by Ca²⁺ imaging. Onincubation of HEK-293 cells with only 400 ng/ml BAY 44-4400, the cellsresponded for 50 minutes in no comparable way with a change in theintracellular Ca²⁺ concentration. However, an effect of BAY 44-4400 canbe found in the presence of α-LTX. Incubation of cells with BAY 44-4400for 6 minutes before addition of 75 nM α-LTX reduces the effect of α-LTXon the Ca²⁺ concentration. The first small increase in [Ca²⁺]_(i)disappears and the second is reduced by 85% 15 minutes after addition ofα-LTX. The response to α-LTX is even stronger if the cells arepreincubated with BAY 44-4400 for 60 minutes before the cells aretreated with FURA for 30 minutes. There is a complete disappearance ofthe first increase in Ca²⁺ and an only very small second increase of 70nM [Ca²⁺]_(i) 30 minutes after addition of 75 nM α-LTX.

In order to show the specific effect of BAY 44-4400 on the action ofα-LTX, two control experiments were carried out. Firstly, HEK-293 cellswere transiently or stably transfected with a mouse β₂-adrenergicreceptor provided with a C-terminal GFP tag, and isoproterenol was usedas ligand.

In fact, isoproterenol also causes a significant Ca²⁺ response:immediately after the addition of isoproterenol there is only oneincrease in Ca²⁺. This increase is, however, unaffected by BAY 44-4400.

In addition, the specificity of the interaction between BAY 44-4400 andHC110-R is shown with the aid of the optical antipode which occupies thesite of BAY 44-4400 as ligand. After preincubation of the cells with 400ng/ml of a PF1022A derivative with 100-fold weaker anthalminticactivity, PF1022-001: cyclo(-L-Lac-D-MeLeu-L-PhLac-D-Me-Leu-)₂ (alsoreferred to as optical antipode) for 90 minutes there was an increase inCa²⁺ of only 110 nM 6 minutes after addition of 75 nM α-LTX, that is tosay a reduction by 59%. When 4 ng/ml of the optical antipode are added 6minutes before the addition of 75 nM α-LTX there is an increase of 67 nM[Ca²⁺]_(i) immediately after addition of the α-LTX. If the concentrationof the optical antipode increases there is a reduction in the increaseto 20 nM Ca²⁺.

The term “polypeptide” as used herein refers both to short amino acidchains, which are usually referred to as peptides, oligopeptides oroligomers, and to longer amino acid chains, which are usually referredto as proteins. It encompasses amino acid chains which may be modifiedeither by natural processes, such as post-translational processing, orby chemical methods, which are state of the art. Such modifications mayoccur at various sites and more than once in a polypeptide, such as, forexample, on the peptide backbone, on the amino acid side chain, at theamino terminus host carboxy terminus. They encompass for exampleacetylations, acylations, ADP ribosylations, amidations, covalentlinkages with flavins, haem portions, nucleotides or nucleotidederivatives, lipids or lipid derivatives or phosphatidylinositol,cyclizations, disulfide bridge formations, demethylations, cystineformations, formylations, gamma-carboxylations, glycosylations,hydroxylations, iodinations, methylations, myristoylations, oxidations,proteolytic processings, phosphorylations, selenoylations andtRNA-mediated additions of amino acids.

The polypeptides may according to the invention be used in the form of“mature” proteins or as parts of larger proteins, e.g. as fusionproteins. They may furthermore have secretions or “leader” sequences,pro-sequences, sequences which make simple purification possible, suchas multiple histidine residues, or additional stabilizing amino acids.

Homologous proteins or polypeptides are regarded as being those proteinsor polypeptides which have at least 70% identity, preferably 80%identity, particularly preferably 90% identity, very particularlypreferably 95% identity, with a sequence as shown in SEQ ID NO: 2 of thedocument DE-A-197 04 024, the contents of which are to be expresslyincluded in the present application, over a length of at least 20,preferably at least 25, particularly preferably at least 30 consecutiveamino acids and very particularly preferably over the complete lengthsthereof.

The polypeptides need not, for their use according to the invention,represent complete receptors, but may also be only fragments thereof aslong as they still have at least the biological activity of the completereceptors. It is moreover unnecessary for the polypeptides to bederivable from transmembrane receptors of H. contortus. Polypeptideswhich correspond to transmembrane receptors of other species ofhelminths or even arthropods, or fragments thereof which are still ableto exercise the biological activity of these receptors, are alsoregarded as being according to the invention.

The polypeptides may, for their use according to the invention, havedeletions or amino acid substitutions compared with the correspondingregion of naturally occurring GPC receptors, as long as they stillexercise at least one biological activity of the complete receptors.Conservative substitutions are preferred. Such conservativesubstitutions encompass variations where one amino acid is replaced byanother amino acid from the following group:

-   1. Small aliphatic, nonpolar or slightly polar residues: Ala, Ser,    Thr, per and Gly;-   2. polar, negatively charged residues and amides thereof: Asp, Asn,    Glu and Gln;-   3. polar, positively charged residues: His, Arg and Lys;-   4. large aliphatic, nonpolar residues: Met, Leu, Ile, Val and Cys;    and-   5. aromatic residues: Phe, Tyr and Trp.

The following list shows preferred conservative substitutions: Originalresidue Substitution Ala Gly, Ser Arg Lys Asn Gln, His Asp Glu Cys SerGln Asn Glu Asp Gly Ala, Pro His Asn, Gln Ile Leu, Val Leu Ile, Val LysArg, Gln, Glu Met Leu, Tyr, Ile Phe Met, Leu, Tyr Ser Thr Thr Ser TrpTyr Tyr Trp, Phe Val Ile, Leu

The sequences described above and in SEQ ID NO: 1 and SEQ ID NO: 3 inDE-A-197 04 024 may additionally be used for finding genes which codefor polypeptides which are involved in the structure of functionallysimilar transmembrane receptors in helminths or arthropods. Functionallysimilar receptors mean according to the present invention receptorswhich encompass polypeptides which, although they differ in the aminoacid sequence from the polypeptides described herein, have essentiallythe same biological function.

The term “essentially the same biological function” as used herein meansinvolvement in the structure of G-protein-coupled heptahelicaltransmembrane receptors capable of functioning, particularly suchreceptors of the sekretin subfamily, or a function corresponding to theHC110-R receptor from H. contortus. Such a function also includes theproperties described above of the receptor, such as the sensitivity toα-LTX as agonist or nifedipine as antagonist of α-LTX.

The term “hybridize” as used herein describes the process in which asingle-stranded nucleic acid molecule undergoes base pairing with acomplementary strand. It is possible in this way, starting from thesequence information disclosed herein, to isolate for example DNAfragments from other nematodes than H. contortus, and from arthropods,which code for polypeptides having the biological activity of GPCreceptors.

In relation to a suitable probe, the amino-terminal and carboxy-terminalcDNA sections are preferred. The hybridization conditions are chosen sothat it is also possible to detect less similar sequences from otherorganisms. The hybridization conditions with reduced stringency may beas follows, for example: hybridization is carried out in 6×SSC/0%formamide as hybridization solution at between 40 and 55° C. Theconditions for the specific 2nd washing step must be tested, e.g.initially 2×SSC at 50° C., then estimation of the signal intensities.The washing conditions are then modified.

Suitable hybridization conditions are indicated by way of example below:

-   Hybridization solution: 6×SSC/0% formamide, preferred hybridization    solution: 6×SSC/25% formamide-   Hybridization temperature: 34° C., preferred hybridization    temperature: 42° C.-   1st washing step: 2×SSC at 40° C.-   2nd washing step: 2×SSC at 45° C.; preferred 2nd washing step:    0.6×SSC at 55° C.;    -   particularly preferred 2nd washing step: 0.3×SSC at 65° C.-   Hybridization conditions are calculated approximately by the    following formula:    The melting temperature Tm=81.5° C.+16.6 log[c(Na⁺)]+0.41(%    G+C))−500/n (Lottspeich and Zorbas 1998).

In this, c is the concentration and n is the length of the hybridizingsequence section in base pairs. The term 500/n is omitted for a sequenceof >100 bp. Maximum stringency washing is at a temperature of 5-15° C.below Tm and an ionic strength of 15 mM Na⁺ (corresponds to 0.1×SSC). Ifan RNA probe is used for the hybridization, the melting point is 10-15°C. higher.

The polypeptides used in the method according to the invention foridentifying compounds with nematicidal and arthropodicidal activity areencoded by the nucleic acids described in SEQ ID NO: 1 and SEQ ID NO: 3in DE-A-197 04 024.

Also included in the use according to the invention are nucleic acidswhich have at least 70% identity, preferably 80% identity, particularlypreferably 90% identity, very particularly preferably 95% identity witha sequence as shown in SEQ ID NO: 1 or SEQ ID NO: 3 over a length of atleast 20, preferably at least 100, particularly preferably at least 500consecutive nucleotides, and very particularly preferably over theentire length thereof.

The nucleic acid according to the invention can likewise be used forproducing transgenic invertebrates. The latter can be employed in testsystems which are based on an expression of the receptors according tothe invention or variants thereof which differs from the wild type. Alsoincluded in this are all transgenic invertebrates for which modificationof other genes or gene control sequences (e.g. promoters) results in achange in the expression of the receptors according to the invention orvariants thereof.

Production of the transgenic invertebrates takes place for example inDrosophila melanogaster by P-element-mediated gene transfer or inCaenorhabditis elegans by transposon-mediated gene transfer (e.g. byTc1, Plasterk 1996).

The invention thus also relates to transgenic invertebrates whichcomprise at least one of the nucleic acids according to the invention,preferably transgenic invertebrates of the species Drosophilamelanogaster or Caenorhabditis elegans, and the transgenic progenythereof. The transgenic invertebrates preferably comprise the receptorsaccording to the invention in a form which differs from the wild type.

The nucleic acid of the invention can be produced in a conventional way.For example, complete chemical synthesis of the nucleic acid molecule ispossible. It is also possible for only short pieces of the sequenceaccording to the invention to be synthesized chemically and for sucholigonucleotides to be labelled radioactively or with a fluorescent dye.The labelled oligonucleotides can be used to screen cDNA librariesproduced starting from nematode mRNA or insect mRNA. Clones whichhybridize to the labelled oligonucleotides are selected for isolation ofthe relevant DNA. After characterization of the isolated DNA, thenucleic acid of the invention is obtained in a simple manner.

The nucleic acid according to the invention can also be produced by PCRmethods using chemically synthesized oligonucleotides.

The term “oligonucleotide(s)” as used herein means DNA molecules whichconsist of 10 to 50 nucleotides, preferably 15 to 30 nucleotides. Theyare, for example, chemically synthesized and can be used as probes.

The nucleic acid according to the invention can be used for theisolation and characterization of the regulatory regions which occurnaturally in the vicinity of the coding region. The present inventionthus likewise relates to such regulatory regions.

The methods according to the invention likewise make use of vectorswhich comprise a nucleic acid to be used according to the invention or aDNA construct to be used according to the invention. Vectors which canbe used are all phages, plasmids, phagemids, phasmids, cosmids, YACs,BACs, artificial chromosomes or particles suitable for particlebombardment, which are used in molecular biology laboratories.

Preferred vectors are pBIN and its derivatives for plant cells, pFL61for yeast cells, pBLUESCRIPT vectors for bacterial cells, lamdaZAP (fromStratagene) for phages.

Various vectors have been used and a plurality of constructs have beenproduced within the scope of the present invention. The presentinvention likewise relates to the vectors used for transient or stabletransformation of cell lines and for stable expression of the HC110-Rreceptor.

EGFP constructs which lead to the expression of fusion proteins with anN-terminal EGFP tag (EGFP-HC110-R) or a C-terminal EGFP tag(HC110-R-EGFP) were produced for transient expression in eukaryoticcells. The present invention likewise relates to the polypeptidesencoded by these vectors and the conventional GFP vectors, and red-shiftand blue-shift variants.

A further construct, the vector pMyc6×His, was used for stableexpression, and the present invention likewise relates thereto. Thevector pMyc6×His is derived from the vector pSecTagA [Invitrogen, Leek,NL] by double digestion with the restriction enzymes NhiI and SfiI,followed by blunting of the ends and religation of the vector.

The present invention also relates to host cells which comprise anucleic acid to be used according to the invention or a vector to beused according to the invention. This invention relates in particular toa stably transformed HEK-293 cell line with HC110-R-Myc/His which hasbeen deposited under the number DSM ACC2464 at the internationaldepositary authority DSMZ-Deutsche Sammlung von Microorganismen undZellkulturen GmbH, Mascheroder Weg lb in 38 124 Braunschweig.

The present invention likewise relates to host cells which comprise anucleic acid to be used according to the invention or a vector to beused according to the invention, and a vector which enables the cells toexpress aequorin, a luminescent protein which 2+emits light in thepresence of Ca²⁺. Corresponding host cells make it possible to followthe change in the Ca²⁺ concentration, and thus the effect of substancesfor example on HC110-R, with the aid of the aequorin indicator. Theinvention relates in particular to the stably transformed HEK-293 cellline with HC110-R-Myc/His which is capable of expressing aequorin andhas been deposited under the number DSM ACC2465 at the internationaldepositary authority DSMZ-Deutsche Sammlung von Microorganismen undZellkulturen GmbH, Mascheroder Weg 1b in 38 124 Braunschweig.

The term “host cell” as used herein refers to cells which do notnaturally contain the nucleic acids according to the invention.

Suitable and preferred host cells are eukaryotic cells such as yeasts,mammalian, amphibian, insect or plant cells. Preferred eukaryotic hostcells are HEK-293, Schneider S2, Spodoptera Sf9, Kc, CHO, HepG-2, K1,COS-1, COS-7, HeLa, C127, 3T3 or BHK cells and, in particular, xenopusoocytes, and HEK-293 or COS-7 cells are very particularly suitable.

This invention likewise relates to the use of DNA corresponding to thesequence ID No. 2 described in the application DE-A-197 04 024 for thedetection of DNA from helminths, preferably from the phylum Nematoda,particularly preferably from the family Trichostrongylidae, veryparticularly preferably of the genus Haemonchus and most preferably ofthe species Haemonchus contortus.

The invention moreover relates to oligonucleotides which correspond to aregion of the DNA sequence described above or its complementary strandand are able to hybridize thereto. The invention relates to the use ofthese oligonucleotides or parts thereof as

-   a) probes in Northern or Southern blot assays,-   b) PCR primers in a diagnostic method for detecting the    above-mentioned nematodes, where the DNA of the relevant helminths    is specifically amplified with the aid of the primers and of the PCR    technique and is then identified.

The invention also relates to a method for detecting helminths,preferably from the phylum Nematoda, particularly preferably from thefamily Trichostrongylidae, very particularly preferably of the genusHaemonchus and most preferably of the species Haemonchus contortus,where oligonucleotides as described above specifically hybridize to DNAsequences which originate from the said organisms, and which are thenamplified with the aid of the PCR technique.

Detection of organisms as mentioned above can take place, for example,by

-   a) providing an oligonucleotide probe or primers which hybridize    onto the DNA coding for HC110-R according to the invention, or    strands complementary thereto, or onto the 5′- or 3′-flanking    regions thereof,-   b) bringing the oligonucleotide probe or the primers into contact    with an appropriately prepared DNA-containing sample,-   c) detecting the hybridization of the oligonucleotide or primer    (e.g. using the polymerase chain reaction),-   d) sequencing the detected sequence of the HC110-R gene, and-   e) comparing the sequence with the DNA sequences according to the    invention, preferably with the sequence ID No. 2 described in the    application DE-A-197 04 024.

The invention therefore likewise relates to a diagnostic test kit fordetecting helminths, preferably from the phylum Nematoda, particularlypreferably from the family Trichostrongylidae, very particularlypreferably of the genus Haemonchus and most preferably of the speciesHaemonchus contortus, which kit makes available inter aliaoligonucleotides as described above which can be used in methods fordetecting species from the systematic groups mentioned.

The invention likewise relates to a diagnostic test kit as describedabove, where the oligonucleotides made available in this test kit areprovided with a detectable marker. Such detectable markers may includeinter alia enzymes, enzyme substrates, coenzymes, enzyme inhibitors,fluorescent markers, chromophores, luminescent markers andradioisotopes.

This invention also relates to the use of the aforementioned HC110-Rpolypeptides or fragments thereof and of receptor proteins which are70%, preferably 80%, particularly preferably 90% and very particularlypreferably 95% homologous thereto from helminths, preferably from thephylum Nematoda, particularly preferably from the familyTrichostrongylidae, very particularly preferably of the genus Haemonchusand most preferably of the species Haemonchus contortus for producingvaccines which comprise at least one HC110-R polypeptide or fragment ora receptor protein thereof which is 70%, preferably 80%, particularlypreferably 90% and very particularly preferably 95% homologous thereto.The vaccine is able in this case to elicit an immune response which isspecific for an HC110-R protein described above.

In a preferred embodiment, the vaccine comprises an antigenicdeterminant, e.g. a single determinant of a polypeptide with an aminoacid sequence according to the sequence ID No. 2 described in theapplication DE-A-197 04 024, or of a polypeptide encoded by theaforementioned DNA or fragments thereof.

The present invention further relates to methods for producing thepolypeptides to be used according to the invention. The polypeptidesencoded by the known nucleic acids can be produced by cultivating hostcells which comprise these nucleic acids under suitable conditions. Thedesired polypeptides can then be isolated from the cells or the culturemedium in a conventional way. The polypeptides can also be produced inin vitro systems.

A rapid method for isolating the polypeptides according to the inventionwhich are synthesized by host cells using a nucleic acid according tothe invention starts for example, as described in the examples, with theexpression of a fusion protein, where the fusion partner can beaffinity-purified in a simple manner. The fusion partner may be, forexample, glutathione S-transferase. The fusion protein can then bepurified on a glutathione affinity column. The fusion partner can beremoved by partial proteolytic cleavage, for example at linkers betweenthe fusion partner and the polypeptide according to the invention whichis to be purified. The linker can be designed so that it includes targetamino acids, such as arginine and lysine residues, which define sitesfor cleavage by trypsin. Such linkers can be generated by employingstandard cloning methods using oligonucleotides. Another possible methodis based on the use of histidine fusion proteins and purificationthereof on Ni²⁺ Talon columns.

Further possible purification methods are based on preparativeelectrophoresis, FPLC, HPLC (e.g. using gel filtration, reverse phase orslightly hydrophobic columns), gel filtration, differentialprecipitation, ion exchange chromatography and affinity chromatography.

Since receptor-like protein kinases are membrane proteins, detergentextractions are preferably carried out in the purification methods, forexample using detergents which affect the secondary and tertiarystructures of the polypeptides only slightly or not at all, such asnonionic detergents.

The purification of the polypeptides to be used according to theinvention may include the isolation of membranes starting from hostcells which express the nucleic acids according to the invention. Suchcells preferably express the polypeptides in a copy number sufficientfor the amount of the polypeptides found in a membrane fraction to be atleast 10 times higher than that found in comparable membranes from cellswhich naturally express the HC110-R gene; the amount is particularlypreferably at least 100 times, very particularly preferably at least1000 times higher.

The terms “isolation or purification” as used herein mean that thepolypeptides are separated from other proteins or other macromoleculesof the cell or the tissue. A composition according to the inventioncontaining the polypeptides is preferably enriched at least 10-fold andparticularly preferably at least 100-fold, in terms of the proteincontent, compared with a preparation from the host cells.

The polypeptides according to the invention can also beaffinity-purified without fusion partners with the aid of antibodieswhich bind to the polypeptides.

The invention further relates to antibodies which bind specifically tothe aforementioned polypeptides or receptors. Such antibodies areproduced in a conventional way. For example, such antibodies can beproduced by injecting a substantially immunocompetent host with anamount which is effective for antibody production of a transmembranereceptor according to the invention, such as the HC110-R receptors fromH. contortus or of a fragment thereof and by subsequent isolation ofthis antibody. It is additionally possible to obtain in a manner knownper se an immortalized cell line which produces monoclonal antibodies.The antibodies may, where appropriate, be labelled with a detectionreagent. Preferred examples of such a detection reagent are enzymes,radiolabelled elements, fluorescent chemicals or biotin. In place of thecomplete antibody it is also possible to employ fragments which have thedesired specific binding properties.

The term “agonist” as used herein refers to a molecule which activatestransmembrane receptors.

The term “antagonist” as used herein refers to a molecule whichdisplaces an agonist from its binding site or inhibits the function ofthe agonist.

The term “modulator” as used herein is generic for agonist andantagonist.

Modulators may be small organic chemical molecules, peptides orantibodies which bind to the polypeptides according to the invention.Modulators may furthermore be small organic chemical molecules, peptidesor antibodies which bind to a molecule which in turn binds to thepolypeptides according to the invention and thus affects theirbiological activity. Modulators may be mimetics of natural substratesand ligands.

The modulators are preferably small organic chemical compounds.

The binding of the modulators to the polypeptides may alter the cellularprocesses in a way leading to the death of the helminths or arthropodstreated therewith.

The present invention therefore also extends to the use of modulators ofthe polypeptides as anthelmintics and arthropodicides.

This invention likewise relates in particular to the use of α-LTX asanthelmintic.

The use according to the invention of the nucleic acids or polypeptidesin a method according to the invention also makes it possible to findcompounds which bind to the receptors according to the invention. Thelatter may likewise be employed as anthelmintics, e.g. as nematicidesfor plants or as anthelmintic active substance for animals. For example,host cells which contain the nucleic acids and express the correspondingreceptors or polypeptides, or the gene products themselves, are broughtinto contact with a compound or a mixture of compounds under conditionswhich allow interaction of at least one compound with the host cells,the receptors or the individual polypeptides.

The present invention relates in particular to a method which issuitable for the identification of nematicidal active substances whichbind to transmembrane receptors from helminths or arthropods, preferablyto GPCR of the sekretin subfamily, particularly preferably to thereceptor HC110-R from H. contortus, and bind to receptors which are 70%,preferably 80%, particularly preferably 90% and very particularlypreferably 95% identical in sequence thereto. The methods may, however,also be carried out with an HC110-R-homologous receptor from a speciesother than the species mentioned here. Methods which use receptors otherthan the HC110-R according to the invention are fully encompassed by thepresent invention.

The methods according to the invention include high throughput screening(e.g. high throughput screening (HTS) and ultra high throughputscreening (UHTS)). It is possible to use for this purpose both hostcells and cell-free preparations which contain the nucleic acidsaccording to the invention host the polypeptides according to theinvention.

Cell-Free Test Systems

Many test systems which aim to test compounds and natural extracts aredesigned for high throughputs in order to maximize the number ofsubstances investigated in a given period. Test systems which are basedon cell-free operations require purified or semipurified protein. Theyare suitable for an “initial” testing which aims primarily to detect apossible effect of a substance on the target protein.

Effects such as cytotoxicity are usually ignored in these in vitrosystems. The test systems moreover examine both inhibitory orsuppressive effects of the substances and stimulatory effects. Theefficacy of a substance can be checked by concentration-dependent testseries. Control mixtures without test substances can be used forassessing the effects.

Cell-Based Test Systems

The development of cell-based test systems for identifying substanceswhich modulate the activity of HC110-R and homologous receptors is madepossible by the cell lines stably transformed with HC110-R which aremade available by the present invention, but also by the correspondinghomologous receptors from other species which can be identified on thebasis of the present invention.

The present invention likewise makes it possible to identify othercompounds which are effective as calcium channel blockers.

Modulators can be found by incubating a synthetic reaction mix (e.g.products of in vitro translation) or a cellular constituent, such as amembrane or any other preparation which contains the polypeptide,together with a labelled substrate or ligand of the polypeptides in thepresence and absence of a candidate molecule which may be an agonist orantagonist. The ability of the candidate molecule to increase or inhibitthe activity of the polypeptides according to the invention becomesevident from an increased or reduced binding of the labelled ligand orfrom an increased or reduced conversion of the labelled substrate.Molecules which bind well and lead to an increased activity of thepolypeptides according to the invention are agonists.

Molecules which bind well but do not induce the biological activity ofthe polypeptides according to the invention are probably goodantagonists.

Scintillation Proximity Assay (SPA)

One possibility for identifying substances which modulate the activityof HC110-R and receptor proteins homologous thereto is the so-calledscintillation proximity assay (SPA), see EP-A-015 473. This test systemmakes use of the interaction of a receptor (e.g. HC110-R) with aradiolabelled ligand (e.g. a small organic molecule or a secondradiolabelled protein molecule). The receptor is in this case bound tomicrospheres or beads which are provided with scintillating molecules.During the decay of radioactivity, the scintillating substance in thesphere is excited by the subatomic particles of the radioactive markerand emits a detectable photon. The test conditions are optimized so thatthe only particles emitted by the ligand which lead to a signal arethose emitted by a ligand bound to the receptor or HC110-R.

In one possible embodiment, HC110-R is bound to the beads, either withor without interacting or binding test substances. It would also bepossible to employ in this case fragments of the HC110-R receptor. Aradiolabelled ligand might be, for example, labelled (α-LTX, nifedipineor a labelled depsipeptide. When a binding ligand binds to theimmobilized HC110-R receptor, this ligand would have to inhibit orabolish an existing interaction between the immobilized HC110-R and thelabelled ligand in order itself to bind in the region of the contactarea. Successful binding to the immobilized HC110-R receptor can then bedetected by means of a flash of light.

Correspondingly, an existing complex between an immobilized and a free,labelled ligand is destroyed by the binding of a test substance, whichleads to a fall in the detected intensity of light flashes. The testsystem then corresponds to a complementary inhibition system.

Two-Hybrid System

Another example of a test system based on whole cells is the so-calledtwo-hybrid system. A specific example thereof is the so-calledinteraction trap. This involves genetic selection of interactingproteins in yeast (see, for example, Gyuris et al. 1993). The testsystem is designed to detect and describe the interaction of twoproteins through successful interaction leading to a detectable signal.

Such a test system can also be adapted for the testing of large numbersof test substances in a given period.

The system is based on the construction of two vectors, the “bait”vector and the “prey” vector. A gene coding for an HC110-R according tothe invention or fragments thereof is cloned into the bait vector andthen expressed as fusion protein with the LexA protein, a DNA-bindingprotein. A second gene coding for an HC110-R interaction partner, forexample for an α-LTX, is cloned into the prey vector where it isexpressed as fusion protein with the B42 prey protein. Both vectors arepresent in a Saccharomyces cerevisiae host which contains copies ofLexA-binding DNA on the 5′ side of a lacZ or HIS3 reporter gene. If aninteraction takes place between the two fusion proteins, transcriptionof the reporter gene is activated. If the presence of a test substanceleads to inhibition or destruction of the interaction, the two fusionproteins are no longer able to interact and the product of the reportergene is no longer produced.

Displacement Test

Another example of a method with which it is possible to find modulatorsof the polypeptides according to the invention is a displacement test inwhich, under conditions suitable for this purpose, the polypeptidesaccording to the invention and a potenial modulator are brought togetherwith a molecule which is known to bind to the polypeptides according tothe invention, such as a natural substrate or ligand or a substrate orligand mimetic. α-LTX is preferably used for this in a manner accordingto the invention.

A known analytical system, e.g. from Biacore AB, Uppsala, Sweden, can beemployed for molecular interaction studies using the complete HC110-Rprotein or the N- host C-terminal deletion mutants of HC110-R, or elsevariants of the HC110-R molecule which have been modified by in vitromutagenesis or other known methods. This may entail on the one hand

-   (i) coupling the HC110-R protein or fragments thereof by known    chemical methods (coupling via amines, thiols, aldehydes) or    affinity binding (e.g. streptavidin-biotin, IMAC) to a biochip, or    on the other hand-   (ii) coupling α-LTX or another modulator, e.g. BAY 44-4400 or other    possible ligands as described under (i) to the chip.

Binding of a ligand (HC110-R protein or any modulator, e.g. BAY 44-4400etc.) present in solution to the immobilized molecules is physicallymeasurable. In the Biacore Instrument, the ligand is immobilized on asensor chip which has a thin layer of gold. The analyte solution isdiffused through a microflow cell on the chip.

Binding of the analyte to the immobilized ligands increases the localconcentration on the surface, with the refractive index of the mediumnear the gold layer gradually increasing. This has an effect on theinteraction between free electrons (plasmons) in the metal and photonswhich are emitted from the instrument. These physical changes areproportional to the mass and number of molecules on the chip, and theligand-analyte binding is recorded in real time, making it possible todetermine the apparent association/dissociation rate (Fivash et al.1998). The specificity of the binding is validated by competitionexperiments.

Corresponding measurements are also used to determine the HC110-Rprotein domains important for the binding of ligands, and foridentifying novel, previously undisclosed ligands of HC110-R.

Calcium Imaging

Calcium imaging or signalling is to be regarded as another method fordetecting substances interacting with HC110-R (see, for example, FIGS.10 and 11). This entails the use of calcium indicators with whose aidchanges in the intracellular calcium level are made detectable.HC110-R-expressing cells which are loaded with calcium indicators areemployed in this method. When there is a calcium influx caused by anHC110-R agonist, or when there is release of intracellular calcium,under UV excitation there is a change in the absorption as a function ofthe calcium loading of the indicator. An antagonist can be identified insuch a system by the complete or partial suppression of the calciumsignal induced by the agonist (e.g. α-LTX). Possible calcium indicatorswhich are suitable for this purpose are Fura-2 (Sigma) or Indo-1(molecular probes).

Further calcium indicators can be excited by visible light and vary intheir fluorescence characteristics in a detectable manner depending ontheir calcium loading. The indicators Fluo-3 and Fluo-4 have a highcalcium affinity. Fluo-4 is suitable, with its stronger fluorescencesignal, in particular for measurements in test systems in which thecells are employed only in low density, as in the case of HEK293 cells.Further indicators are Rhod-2, x-Rhod-1, Fluo-5N, Fluo-5F, Mag-Fluo-4,Rhod-5F, Rhod-5N, Y-Rhod-5N, Mag-Rhod-2, Mag-X-Rhod-1, Calcium Green-1and -2, Calcium Green-5N, Oregon Green 488 BAPTA-1, Oregon Green 488BAPTA-2 and −5N, Fura Red, calcein and the like.

An alternative to loading cells with calcium indicators is recombinantexpression of photoproteins in the target cells. These photoproteinsthen respond in the form of light emission after they have formed acomplex with calcium ions. A photoprotein which has already been usedmany times in numerous investigations and test systems is aequorin. Thecells expressing the target protein and simultaneously aequorin arefirst loaded with the luminophor coelenterazine in this test method. Theapoaequorin formed by the cells forms a complex with the coelenterazineand carbon dioxide. If calcium subsequently also enters the cells andbinds to the complex there is release of carbon dioxide and blue light(emissions maximum ˜466 nm). The light emission in this case correlateswith the calcium concentration prevailing inside the cell.

This invention thus likewise relates to the use of HC110-R, fragmentsthereof or similar proteins from other organisms in a test method inwhich the function of the receptor or the binding of modulators to thereceptor is detected by means of a light signal mediated by aequorin orsimilar photoproteins.

It is also possible in this way by using host cells or transgenicinvertebrates which comprise the nucleic acid according to the inventionto find substances which alter the expression of the receptors. Suchsubstances may also represent valuable anthelmintics.

The active substances found with the aid of the method according to theinvention are correspondingly suitable for controlling animal pests, inparticular insects, arachnids and nematodes which occur in agriculture,in forests, in the storage and protection of materials, and in thehygiene sector. The active substances found by the method according tothe invention are particularly suitable for controlling nematodes andarachnids. The abovementioned pests include:

-   From the order of Isopoda e.g. Oniscus asellus, Annadillidium    vulgare, Porcellio scaber.-   From the order of Diplopoda e.g. Blaniulus guttulatus.-   From the order of Chilopoda e.g. Geophilus carpophagus, Scutigera    spp.-   From the order of Symphyla e.g. Scutigerella immaculata.-   From the order of Thysanura e.g. Lepisma saccharina.-   From the order of Collembola e.g. Onychiurus ammatus.-   From the order of Orthoptera e.g. Acheta domesticus, Gryllotalpa    spp., Locusta migratoria migratorioides, Melanoplus spp.,    Schistocerca gregaria.-   From the order of Blattaria e.g. Blatta orientalis, Periplaneta    americana, Leucophaea maderae, Blattella gernanica.-   From the order of Dermaptera e.g. Forficula auricularia.-   From the order of Isoptera e.g. Reticulitermes spp.-   From the order of Phthiraptera e.g. Pediculus humanus corporis,    Haematopinus spp., Linognathus spp., Trichodectes spp., Damalinia    spp.-   From the order of Thysanoptera e.g. Hercinothrips femoralis, Thrips    tabaci, Thrips palmi, Franklinella accidentalis.-   From the order of Heteroptera e.g. Eurygaster spp., Dysdercus    intermedius, Piesma quadrata, Cimex lectularius, Rhodnius prolixus,    Triatoma spp.-   From the order of Homoptera e.g. Aleurodes brassicae, Bemisia    tabaci, Trialeurodes vaporariorum, Aphis gossypii, Brevicoryne    brassicae, Cryptomyzus ribis, Aphis fabae, Aphis pomi, Eriosoma    lanigerum, Hyalopterus arundinis, Phylloxera vastatrix, Pemphigus    spp., Macrosiphum avenae, Myzus spp., Phorodon humuli, Rhopalosiphum    padi, Empoasca spp., Euscelis bilobatus, Nephotettix cincticeps,    Lecanium corni, Saissetia oleae, Laodelphax striatellus, Nilaparvata    lugens, Aonidiella aurantii, Aspidiotus hederae, Pseudococcus spp.,    Psylla spp.-   From the order of Lepidoptera e.g. Pectinophora gossypiella, Bupalus    piniarius, Cheimatobia brumata, Lithocolletis blancardella,    Hyponomeuta padella, Plutella xylostella, Malacosoma neustria,    Euproctis chrysorrhoea, Lymantria spp., Bucculatrix thurberiella,    Phyllocnistis citrella, Agrotis spp., Euxoa spp., Feltia spp.,    Earias insulana, Heliothis spp., Mamestra brassicae, Panolis    flammea, Spodoptera spp., Trichoplusia ni, Carpocapsa pomonella,    Pieris spp., Chilo spp., Pyrausta nubilalis, Ephestia kuehniella,    Galleria mellonella, Tineola bisselliella, Tinea pellionella,    Hofmannophila pseudospretella, Cacoecia podana, Capua reticulana,    Choristoneura fumiferana, Clysia ambiguella, Homona magnanima,    Tortrix viridana, Cnaphalocerus spp., Oulema oryzae.-   From the order of Coleoptera e.g. Anobium punctatum, Rhizopertha    dominica, Bruchidius obtectus, Acanthoscelides obtectus, Hylotrupes    bajulus, Agelastica alni, Leptinotarsa decemlineata, Phaedon    cochleariae, Diabrotica spp., Psylliodes chrysocephala, Epilachna    varivestis, Atomaria spp., Oryzaephilus surinaniensis, Anthonomus    spp., Sitophilus spp., Otiorrhynchus sulcatus, Cosmopolites    sordidus, Ceuthorrhynchus assimilis, Hypera postica, Dermestes spp.,    Trogoderma spp., Anthrenus spp., Attagenus spp., Lyctus spp.,    Meligethes aeneus, Ptinus spp., Niptus hololeucus, Gibbium    psylloides, Tribolium spp., Tenebrio molitor, Agriotes spp.,    Conoderus spp., Melolontha melolontha, Amphimallon solstitialis,    Costelytra zealandica, Lissorhoptrus oryzophilus.-   From the order of Hymenoptera e.g. Diprion spp., Hoplocampa spp.,    Lasius spp., Monomorium pharaonis, Vespa spp.-   From the order of Diptera e.g. Aedes spp., Anopheles spp., Culex    spp., Drosophila melanogaster, Musca spp., Fannia spp., Calliphora    erythrocephala, Lucilia spp., Chrysomyia spp., Cuterebra spp.,    Gastrophilus spp., Hyppobosca spp., Stomoxys spp., Oestrus spp.,    Hypoderma spp., Tabanus spp., Tannia spp., Bibio hortulanus,    Oscinella frit, Phorbia spp., Pegomyia hyoscyami, Ceratitis    capitata, Dacus oleae, Tipula paludosa, Hylemyia spp., Liriomyza    spp.-   From the order of Siphonaptera e.g. Xenopsylla cheopis,    Ceratophyllus spp.-   From the class of Arachnida e.g. Scorpio maurus, Latrodectus    mactans, Acarus siro, Argas spp., Ornithodoros spp., Dermanyssus    gallinae, Eriophyes ribis, Phyllocoptruta oleivora, Boophilus spp.,    Rhipicephalus spp., Amblyomma spp., Hyalomma spp., Ixodes spp.,    Psoroptes spp., Chorioptes spp., Sarcoptes spp., Tarsonemus spp.,    Bryobia praetiosa, Panonychus spp., Tetranychus spp., Hemitarsonemus    spp., Brevipalpus spp.

The plant-parasitic nematodes include e.g. Pratylenchus spp., Radopholussimilis, Ditylenchus dipsaci, Tylenchulus semipenetrans, Heteroderaspp., Globodera spp., Meloidogyne spp., Aphelenchoides spp., Longidorusspp., Xiphinema spp., Trichodorus spp., Bursaphelenchus spp.

The active substances found using the method according to the inventionare active not only against plant, hygiene and stored product pests, butalso in the veterinary medical sector against animal parasites(ectoparasites) such as hard ticks, soft ticks, mange mites, leaf mites,flies (biting and licking), parasitic fly larvae, lice, hair lice, furlice and fleas. These parasites include:

-   From the order of Anoplurida e.g. Haematopinus spp., Linognathus    spp., Pediculus spp., Phtirus spp., Solenopotes spp.-   From the order of Mallophagida and the suborders Amblycerina and    Ischnocerina e.g. Trimenopon spp., Menopon spp., Trinoton spp.,    Bovicola spp., Werneckiella spp., Lepikentron spp., Damalina spp.,    Trichodectes spp., Felicola spp.-   From the order Diptera and the suborders Nematocerina and    Brachycerina e.g. Aedes spp., Anopheles spp., Culex spp., Simulium    spp., Eusimulium spp., Phlebotomus spp., Lutzomyia spp., Culicoides    spp., Chrysops spp., Hybomitra spp., Atylotus spp., Tabanus spp.,    Haematopota spp., Philipomyia spp., Braula spp., Musca spp.,    Hydrotaea spp., Stomoxys spp., Haematobia spp., Morellia spp.,    Fannia spp., Glossina spp., Calliphora spp., Lucilia spp.,    Chrysomyia spp., Wohlfahrtia spp., Sarcophaga spp., Oestrus spp.,    Hypoderma spp., Gasterophilus spp., Hippobosca spp., Lipoptena spp.,    Melophagus spp.-   From the order of Siphonapterida e.g. Pulex spp., Ctenocephalides    spp., Xenopsylla spp., Ceratophyllus spp.-   From the order of Heteropterida e.g. Cimex spp., Triatoma spp.,    Rhodnius spp., Panstrongylus spp.-   From the order of Blattarida e.g. Blatta orientalis, Periplaneta    americana, Blattela germanica, Supella spp.-   From the subclass of Acaria (Acarida) and the orders of Meta- and    Mesostigmata e.g. Argas spp., Ornithodorus spp., Otobius spp.,    Ixodes spp., Amblyomma spp., Boophilus spp., Dennacentor spp.,    Haemophysalis spp., Hyalomma spp., Rhipicephalus spp., Dennanyssus    spp., Raillietia spp., Pneumonyssus spp., Sternostoma spp., Varroa    spp.-   From the order of Actinedida (Prostigmata) and Acaridida (Astigmata)    e.g. Acarapis spp., Cheyletiella spp., Ornithocheyletia spp., Myobia    spp., Psorergates spp., Demodex spp., Trombicula spp., Listrophorus    spp., Acarus spp., Tyrophagus spp., Caloglyphus spp., Hypodectes    spp., Pterolichus spp., Psoroptes spp., Chorioptes spp., Otodectes    spp., Sarcoptes spp., Notoedres spp., Knemidocoptes spp., Cytodectes    spp., Laminosioptes spp.

The active substances found with the aid of the method of the inventionare also suitable for controlling mites, especially house dust mites,e.g. Dermatophagoides pteronyssinus and D. farinae.

The active substances found using the method according to the inventionare also suitable for controlling arthropods which infest agriculturalproductive livestock such as, for example, cattle, sheep, goats, horses,pigs, donkeys, camels, buffaloes, rabbits, chickens, turkeys, ducks,geese, bees, other pets such as, for example, dogs, cats, caged birdsand aquarium fish, and so-called experimental animals such as, forexample, hamsters, guinea pigs, rats and mice. Control of thesearthropods is intended to reduce deaths and decreases in production (ofmeat, milk, wool, hides, eggs, honey etc.) so that more economic andeasier animal husbandry is possible through use of the active substancesaccording to the invention.

Compounds found with the aid of the described methods and polypeptidesare likewise valuable for the treatment of animals and humans infectedby pathogenic endoparasites of humans or of productive livestock, pets,zoo animals and laboratory and experimental animals.

The compounds can be used at all stages of development of normal,sensitive strains and resistance strains. It is possible by treatmentwith compositions which contain one or more of these compounds both toprevent economic losses in productive livestock and treat diseases inhumans and animals. The following parasites are of particular interestas targets of the active substances found:

-   Enoplida, e.g. Trichuris spp., Capillaria spp., Trichomosoides spp.,    Trichinella spp.-   Rhabditia, e.g. Micronema spp., Strongyloides spp.-   Strongylida, e.g. Strongylus spp., Triodontophorus spp.,    Oesophagodontus spp., Trichonema spp., Gyalocephalus spp.,    Cylindropharynx spp., Poteriostomum spp., Cyclococercus spp.,    Cylicostephanus spp., Oesophagostomum spp., Chabertia spp.,    Stephanurus spp., Ancylostoma spp., Uncinaria spp., Bunostomum spp.,    Globocephalus spp., Syngamus spp., Cyathostomum spp., Cylicocyclus    spp., Neostrongylus spp., Cystocaulus spp., Pneumostrongylus spp.,    Spicocaulus spp., Elaphostrongylus spp., Parelaphostrongylus spp.,    Crenosoma spp., Paracrenosoma spp., Angiostrongylus spp.,    Aelurostrongylus spp., Filaroides spp., Parafilaroides spp.,    Trichostrongylus spp., Haemonchus spp., Ostertagia spp.,    Marshallagia spp., Cooperia spp., Nematodirus spp., Hyostrongylus    spp., Obeliscoides spp., Amidostomum spp., Ollulanus spp.    Dictyocaulus spp., Muellerius spp., Protostrongylus spp.-   Oxyurida, e.g. Oxyuris spp., Enterobius spp., Passalurus spp.,    Syphacia spp., Aspi-culuris spp., Heterakis spp.-   Ascaridia, e.g. Ascaris spp., Toxascaris spp., Toxocara spp.,    Parascaris spp., Anisa-kis spp., Ascaridia spp.-   Spirurida, e.g. Gnathostoma spp., Physaloptera spp., Thelazia spp.,    Gongylonema spp., Habronema spp., Parabronema spp., Draschia spp.,    Dracunculus spp.-   Filariida, e.g. Stephanofilaria spp., Parafilaria spp., Setaria    spp., Loa spp., Dirofilaria spp., Litomosoides spp., Brugia spp.,    Wuchereria spp., Onchocerca spp.-   Gigantorhynchida, e.g. Filicollis spp., Moniliformis spp.,    Macracanthorhynchus spp., Prosthenorchis spp.-   Mastigophora (Flagellata)-   Trypanosomatidae, e.g. Trypanosoma b. brucei, T. b. gambiense, T. b.    rhodesiense, T. congolense, T. cruzi, T. evansi, T. equinum, T.    lewisi, T. percae, T. simiae, T. vivax, Leishmania brasiliensis, L.    donovani, L. tropica-   Trichomonadidae, e.g. Giardia lambilia, G. canis.-   Sarcomastigophora (Rhizopoda), e.g. Entamoeba histolytica-   Hartmanellidae, e.g. Acanthamoeba sp., Hartmanella spp.-   Apicomplexa (Sporozoa), e.g. Eimeria acervulina, E. adenoides, E.    alabahmensis, E. anatis, E. anseris, E. arloingi, E. ashata, E.    auburnensis, E. bovis, E. brunetti, E. canis, E. chinchillae, E.    clupearum, E. columbae, E. contorta, E. crandalis, E. debliecki, E.    dispersa, E. ellipsoidales, E. falciformis, E. faurei, E.    labbeana, E. leucarti, E. magna, E. maxima, E. media, E.    meleagridis. E. meleagrimitis, E. mitis, E. necatrix, E.    ninakohlyakimovae, E. ovis, E. parva, E. pavonis, E. perforans, E.    phasani, E. piriformis, E. praecox, E. residua, E. scabra, E.    spec., E. stiedai, E. suis, E. tenella, E. truncata, E. truttae, E.    zuernii, Globidium spec., Isospora belli, I. canis, I. felis, I.    ohioensis, I. rivolta, I. spec., I. suis, Neospora caninum,    Cystisospora spec. Cryptosporidium spec.-   Toxoplasmadidae, e.g. Toxoplasma gondii-   Sarcocystidae, e.g. Sarcocystis bovicanis, S. bovihominis, S.    neuvona, S. ovicanis, S. ovifelis, S. spec., S. suihominis-   Leucozoide, e.g. Leucozytozoon simondi-   Plasmodiidae, e.g. Plasmodium berghei, P. falciparum, P.    malariae, P. ovale, P. vivax, P. spec.-   Piroplasmea, e.g. Babesia argentina, B. bovis, B. canis, B. spec.,    Theileria parva, T. spec.-   Adeleina, e.g. Hepatozoon canis, H. spec.    Also of importance are-   Myxospora and Microspora, e.g. Glugea spec. and Nosema spec., and    Pneumocystis carinii, Ciliophora (Ciliata), e.g. Balantidium coli,    Ichthiophthirius spec., Trichondina spec. or Epistylis spec.

The compounds and compositions found are likewise effective for protozoaof insects such as those of the phylum Microsporidia, particularly thoseof the order Nosema, very particularly those of the species Nosema apis,which are parasites of honey bees.

The present invention therefore also relates to the use of compoundswhich have been found using a method according to the invention hostusing the nucleic acids or polypeptides according to the invention forproducing a composition for controlling helminths host arthropods.

EXAMPLES

1. Construction and Screening of the cDNA Library

The construction of the cDNA library, the production of KLH-PF1022Aconjugate and antiserum against PF1022A, and the immunoscreening of thecDNA library and the DNA analysis took place as described in WO98/15625.

2. Isolation of RNA

The total RNA was extracted and isolated from adult Haemonchus contortusnematodes by the GTC/CsCl cushion method (Sambrook et al. 1989) orobtained by a single step by GTC/phenol/chloroform extraction(Chomczynski and Sacchi 1987). Poly (A)⁺ RNA was isolated bychromatography on oligo(dT)cellulose (Aviv and Leder, 1972).

3. Northern Blotting

Glyoxylated total RNA from Haemonchus contortus (20 μg pe lane) werefractionated in an agarose gel (Sambrook et al. 1989; McMaster andCarmichael 1977) and transferred to a Hybond N membrane (Amersham) bythe basic capillary transfer method (Chomczynski, 1992). Radiolabelledprobes were prepared by randomized labelling of linearized plasmid DNA(HC110-R) using a Megaprime kit (Amersham, Braunschweig, Germany) and 50μCi of [α-³²P]dCTP (3000 Ci/mmol, ICN, Meckenheim, Germany). Thehybridization was carried out in 6×SSC (1×SSC:0.15 M NaCl, 0.015 M Nacitrate), 5× Denhardt's reagent (0.1% polyvinylpyrrolidone, 0.1% BSA,0.1% Ficoll 400), 0.1% SDS and 100 μg/ml herring sperm DNA at 65° C.overnight. The filters were washed with high stringency in 0.1×SSC and0.1% SDS at 65° C. and exposed (Kodak BioMax MS films at −80° C.), seeFIG. 1(A).

4. 5′- and 3′ RACE PCR

The 5′/3′ RACE method was used to isolate the 5′ and 3′ ends missing inthe identified cDNA clone.

The 5′ RACE is based on specific amplification of the 5′ end of a genefrom mRNA. A sequence-specific primer and AMV reversed transcriptase areused to synthesize the first cDNA strand. A poly (A) tail is attached tothe product, so that it is possible to employ an oligo dT anchor primerand a nested sequence-specific primer in the subsequent PCR. It ispossible in a second PCR to use another nested primer in order to ensurespecifity.

In the 3′ RACE, the first cDNA strand is synthesized using an oligo dTprimer and the subsequent PCR reactions take place withsequence-specific primers.

cDNA was synthesized from 1 μg of total H. contortus RNA using theprimer 5′-GGT CAC CGT CGT CCC AGA AA-3′ and the 5′ RACE kit from GIBCOBRL (Eggenstein, Germany). The Superscript reverse transcriptase wasused for this purpose. C tailing of the C terminus of the cDNA wascarried out using terminal deoxynucleotidyltransferase. The firstamplification took place with 400 nM of an oligodeoxyinosyl anchorprimer (5′-CUA CUA CUA CUA GGC CAC GCG TCG ACT AGT ACG GGI IGG GII GGGIIG-3′) and 400 nM of the first nested primer 5′-CCA TTC GAT TCC TCT TCTCG-3′ (Birsner und Grob, Denzlingen, Germany) in 50 μl containing 200 μMof each dNTP, 1.5 mM MgCl₂, a fifth of the tailed cDNA and 2.5 U ofnative Taq polymerase. The first denaturation took place at 94° C. for 5minutes, followed by 35 cycles each of 1 minute at 94° C., 1 minute at53° C. and 2 minutes at 72° C., in turn followed by a last synthesisstep of 10 minutes at 72° C. The reaction conditions for the nested PCRwere the same as described above with the exception that 1 μl of thefirst amplification product was used as template, and the gene-specificsecond nested primer 5′-GTC GAT GGT GCA GAT TTC GC-3′, a truncated formof the anchor primer (5′-CUA CUA CUA CUA GGC CAC GCG TCG ACT AGT AC-3′)and only 25 cycles were carried out at an annealing temperature of 53°C.

The 3′ RACE PCR (GIBCO BRL, Eggenstein, Germany) was carried out with 1μg of the total RNA from adult H. contortus and 500 nM of the oligo dTadapter primer 5′-GGC CAC GCG TCG ACT AGT ACT TTT TTT TTT TTT TTT T-3′,starting with a preincubation at 70° C. for 10 minutes and at 4° C. for2 minutes. After addition of 2.5 mM MgCl₂, 500 μM of each dNTP, 10 μMDTT and 200 U of Super-Script II reverse transcriptase in 20 μl, thecDNA was incubated at 42° C. for 50 minutes and, in a final step, at 70°C. for 15 minutes and at 4° C. for 10 minutes. The RNA was removed by 2U of E. coli RNase H and incubation at 37° C. for 10 minutes. The firstamplification took place with 400 nM of a universal adapter primer(5′-CUA CUA CUA CUA GGC CAC GCG TCG ACT AGT AC-3′) and 400 nM of thesequence-specific primer 5′-TTTGTTCTT CCT TOG TAT CC-3′ in 50 μLcontaining 200 mM of each dNTP, 1.5 mM MgCl₂, a tenth of the tailed cDNAand 2.5 U of native Taq DNA polymerase. The first denaturation step tookplace at 94° C. for 3 minutes, followed by 35 cycles each of 1 minute at94° C., 1 minute at 51° C. and 2 minutes at 72° C., and a finalsynthesis step of 15 minutes at 72° C. 2 μl of the first amplificationwere used for the nested PCR, with the conditions otherwise identical,but now using the shorter adapter primer 5′-GGC CAC GCG TCG ACT AGTAC-3′ and the nested primer 5′-ACA CTC TAA TCT CCA ACT G-3′ with 30cycles in this case. The PCR products were analyzed on 2% agarose gels,eluted and cloned into the TA vector pMosBlue (Amersham, Braunschweig,Germany).

5. Transfer of the RNA

Transfer of the fractionated RNA from the glyoxal gel and of genomic DNAfrom a TBE agarose gel to a neutral Hybond N nylon membrane [Amersham,Braunschweig] takes place by the downward alkaline capillary transfer inalkaline transfer solution by the method of Chomczynski (1992) for 2 h.The membrane is then neutralized in 0.2 M sodium phosphate buffer (pH6.8) for 20 min, dried and baked at 80° C. for 20-60 min. For thehybridization with radiolabelled probes, the membrane is sealed with 5ml/cm² prehybridization solution in plastic bags and incubated at 65° C.for 3 h. The buffer is replaced by hybridization solution for thehybridization. On use of Rapid Hyb. solution [Amersham, Braunschweig],the prehybridization takes place at 65° C. without additionalprehybridization solution in 30 min. After addition of the proberadiolabelled by random priming, the hybridization takes place at 65° C.overnight.

The membrane is then washed once with 2×SSC/0.1% SDS at 65° C. for 20min and three times with 0.1×SSC/0.1% SDS for 1 h each time. Themembrane is exposed to Kodak X-OMAT or Kodak BIOMAX-MS X-ray films withappropriate intensifying screen at −80° C.

6. In vitro Transcription and Translation

The TNT T7/T3-coupled reticulocyte lysate system (Promega) was used totranscribe and translate the full length of the coding sequence ofHC110-R in the presence of ³⁵S-labelled methionine and cysteine (ICN,Eschwege, Germany) in accordance with the manufacturer's protocols. TheRNA was translated using a rabbit reticulocyte lysate system (Promega,Serva, Heidelberg) using a 40 μCi ³⁵S-labelled mixture (>1000 Ci/mmol,ICN, Meckenheim, Germany), 1 μg of circularized HC110-R plasmid DNA and10 U of T3 RNA polymerase. The reaction was carried out at 30° C. for 90minutes. A positive control contained 1 μg of luciferase control DNA.

The reaction products were fractionated by SDS polyacrylamide gelelectrophoresis (Lämmli 1970) and fluorographed using the amplifyfluorography solution (Amersham Pharmacia Biotech) or 1 M sodiumsalicylate (pH 7) (Chamberlain 1979), and the gels were then dried andexposed (Kodak BioMax MR film with intensifier at −80° C.), see FIG.1(B).

Alternatively, the MAXIscript In Vitro Transcription Kit from Ambion(Heidelberg) was used for the in vitro synthesis of an RNA transcriptfrom HC110-R cDNA cloned into pBluescript SK. 4 μg of HC110-R plasmidDNA were linearized with the restriction enzyme Hind III or Sal I behindthe stop codon, extracted with phenol/chloroform, precipitated in 3 vol.of ethanol and dissolved in DEPC/H₂O. 2 μg of HC110-R cDNA in a 50 μlreaction mixture were mixed with 2.5 μl of 200 mM DTT, 2.5 μl each of 10mM ATP, CTP, GTP and UTP, 5 μl of 10× transcription buffer, 1.6 μl ofRNasin inhibitor (40 μl) (Promega, Heidelberg) and 2 μl of T3 phagepolymerase (10 U/μl) and incubated at 37° C. for 2 h. After 1 h, afurther 2 μl of T3 phage polymerase (10 U/μl) were added. The mixturewas mixed with 1.5 μl of RNase-free DNase I (2 U/μl) and 1 μl of RNasininhibitor (40 U/μl), incubated at 37° C. for 15 min, extracted withphenol/chloroform, precipitated in 3 vol. of ethanol and resuspended in25 μl of DEPC/H₂O. ⅕ of the volume of the sample was analyzed in aglyoxal gel.

7. DNA Analysis

The clones were sequenced by the dideoxynucleotide chain terminationmethod with the assistance of automated laser fluorescence sequencing(LICOR 4000; MWG, Ebersberg, Germany), and the Thermo Sequenasefluorescent-labelled cycle sequencing kit (Amersham, Braunschweig,Germany). The sequences of both strands were determined, and thesequence data were [lacuna] with the GCG software (Genetics ComputerGroup Inc., Madison, Wis., USA) and the PC/GENE software(Intelligenetics, Mountain View, Calif., USA). The programmes FASTA(Pearson and Lipman 1988), BLITZ (Smith and Waterman 1981) and BLAST(Altschul and Lipmann 1990) were used to screen the EMBL and Swiss-Protprotein databases for sequence similarities of the derived proteinsequence. The protein sequences were analyzed using SAPS (Brendel et al.1992) and using PROSITE and Prot-Param (Appel et al. 1994).

8. Preparation of Protein Extracts from Haemonchus contortus

50-100 mg of H. contortus worms frozen in liquid nitrogen were taken upin 1 ml of TRIZOL [Gibco, Karlsruhe]. The nematodes were homogenizedtogether with the TRIZOL in a glass potter for 3×15 sec and incubated atRT for 5 min. After addition of 200 μl of chloroform per ml of TRIZOLand shaking of the sample for 15 sec, the mixture was incubated at RTfor a further 2-3 min, before it was centrifuged at 7 000-12 000 rpm and4° C. for 10 min. The upper aqueous phase contains the RNA, theinterphase the genomic DNA and the red organic phase the proteins(Coombs et al. 1990; Chomczynski 1993). The aqueous phase was removedand worked up separately. The interphase and organic phase were mixedwith 300 μl of 100% ethanol per ml of TRIZOL, thoroughly mixed andincubated at RT for 2-3 min. After centrifugation at 2 000 rpm and 4° C.for 5 min, the protein supernatant was cautiously transferred into a newEppendorf vessel and precipitated with 1 ml of isopropanol at RT for 10min. Centrifugation was again carried out at 12 000 rpm and 4° C. for 10min, the supernatant was discarded, and the protein pellet was mixedwith 2 ml portions of 0.3 M guanidine hydrochloride solution in 95%ethanol, vortexed, incubated at RT for 20 min and centrifuged at 7 500rpm and 4° C. for 5 min, 3 times.

The pellet was then dissolved in 2 ml of 100% ethanol, precipitated atRT for 20 min and pelleted at 7 500 rpm and 4° C. for 5 min. The pelletwas briefly dried in vacuo and resuspended in urea lysis buffer (8 M).Insoluble material was removed by centrifugation at 10 000 rpm and 4° C.for 10 min, and the supernatant was transferred into a new Eppendorfvessel and, after determination of the protein concentration, stored at−20° C. until processed further.

9. Preparation of Protein Extracts from Yeast Cells

Protein extracts were prepared from a yeast culture by inoculating 5 mlof YPAD medium with a single yeast colony and shaking at 30° C. untilsaturated (2 days). 2 ml portions of such a yeast culture werecentrifuged at 3 000 rpm and 4° C. (Heraeus Biofuge 15 R) for 2 min,washed once with H₂O, resuspended in 250 μl of yeast lysis buffer,transferred into a small test tube, charged with glass beads (Ø 0.5 mm)up to just below the liquid level and vortexed at the highest settingfor 5 min. Then 4× RotiLoad buffer (Roth, Karlsruhe) was added, and themixture was incubated at 95° C. for 5 min. The disrupted cells weretransferred into an Eppendorf vessel, and the cell detritus was removedby centrifugation at 14 000 rpm (Eppendorf Centrifuge 5415 C) for 5 min.20 μl portions of the samples prepared in this way were loaded onto anSDS polyacrylamide gel.

10. Preparation of Protein Extracts from E. coli Cells

Protein extracts were prepared by pelleting 1×10⁷ E. coli cells from a 5ml overnight culture at 5 000 rpm and 4° C. in a Heraeus floorcentrifuge for 5 min, washed with 5 ml of PBS and again centrifuged. Thepellet was then resuspended in 1 ml of TRIZOL (Gibco, Karlsruhe) andprocessed further.

As an alternative to this, the PBS-washed cell pellet was resuspended inPBS again and the cells were broken up by means of several shortultrasound pulses (Sonifier B-12, Branson Sonic Power Company, Danbury,U.S.A.), liquid nitrogen and by addition of lysozyme.

Following the protein determination, 4×RotiLoad buffer (Roth, Karlsruhe)was added and the fraction was analyzed by loading onto an SDSpolyacrylamide gel. The total amount of protein loaded per lane was10-20 μg.

11. Preparation of Protein Extracts from Cell Culture

A confluent 35 mm cell culture dish of adherent cells or 5-10×10⁶ nonadherent mammalian cells in FCS-containing medium were previouslyremoved by trypsinization with a trypsin/EDTA solution or detachedmechanically after exposure to cold using a cell scraper, transferredinto an Eppendorf vessel, pelleted at 13 000 rpm and RT for 10 sec,washed twice with PBS and again centrifuged. After the cells had beentaken up in 1 ml of TRIZOL they were lysed by vigorous vortexing or bydrawing the cells several times through the needle of a disposablesyringe, and incubated at RT for 5 min. As an alternative to this it isalso possible for the cell pellet to be resuspended in 1 ml of PBS orurea lysis buffer (8 M) and subjected to a very brief ultrasoundtreatment (Sonifier B-12, Branson Sonic Power Company, Danbury, U.S.A.).The protein content is then determined by the method of Bradford (1976)or Lowry et al. (1951).

12. Inducible Protein Expression in E. coli

Polyclonal antibodies against HC110-R protein were produced by threeHC110-R fragments—the complete HC110-R cDNA, the N-terminal end withoutTM domains and the C-terminal end after the 7th TM domain—being clonedinto the expression vector pRSET B (Invitrogen, Leek, NL) and thus fusedN-terminally to a 6×His tag while complying with the reading frame.

The complete coding region of HC110-R was amplified using the P84_ATGBamHI 5′ primer 5′-CTG CCG GAT CCT CGG TTT AAT ACC AAC ATG AGG-3′ andthe P3121_TGA HindIII 3′ primer 5′-GCA CTA AGC TTG ACT GAA GCG CAC AACCTC G-3′, the N terminus up to the 1st transmembrane domain wasamplified using the P84_ATG BamHI 5′ primer (see above) and theP1434_TGA HindIII 3′ primer 5′-GGC TCA AGC TTA TCA GAG AAC AAG CGA CACGGC-3′, and the C terminus starting after the 7th transmembrane domainwas amplified using the P2486_ATG BamHI 5′ primer 5′-CTA TCG GAT CCC AACATG GCT GGC TCC CGT GAT ACC TCT AGG-3′ and the P3121_TGA HindIII 3′primer (see above). The annealing temperature for all three plasmid PCRswas initially 56° C. for 5 cycles and then 62° C. for 30 cycles. The PCRproducts were digested with the enzymes BamHI and HindIII and ligated ina directed manner into the pRSET B expression vector which had likewisebeen linearized with BamHI/HindIII.

In the pRSET B vector, expression is controlled by a viral promoter ofbacteriophages T7. The cloning therefore took place in the XL1-Blue E.coli strain which contains no T7 RNA polymerase gene. The recombinantplasmid was then transformed into T7 polymerase-expressingBL21(De3)pLysS E. coli cells which additionally contain the plasmidpACYC184 which is stabilized by a chloramphenicol-resistence, and whichexpress small amounts of the T7 lysozyme, a natural inhibitor of T7 RNApolymerase. Since these cells are under the control of the lacUV5promoter, IPTG induction leads to expression of the T7 RNA polymeraseand thus also to that of the fusion protein.

A single colony with the required HC110-R plasmid was transferred from afresh LB plate with 50 Lg/ml ampicillin and 35 μg/ml chloramphenicolinto 50 ml of LB medium under the same selection pressure and shaken at280 rpm and 37° C. overnight up to the stationary phase. The overnightculture was then diluted to OD₆₀₀=0.3, and 100 ml of the culture wereshaken further at 280 rpm and 37° C. until OD₆₀₀=0.6-0.5.

1 OD₆₀₀ unit was removed, and the uninduced cells were brieflycentrifuged and taken up in 150 μl of 8 M urea lysis buffer, pH 8.0, and50 μl of 4×RotiLoad buffer [Roth, Karlsruhe]. The uninduced sample wasdenatured for 2 min, the genomic DNA was sheared by short ultrasoundpulses of a few seconds in a Sonifier B-12 [Branson Sonic Power Company,Danbury, U.S.A.], and insoluble particles were pelleted bycentrifugation at 14 000 rpm for 3 minutes.

Expression of the fusion protein was induced by adding 1 mM IPTG, andthe 100 ml of culture were incubated at 37° C. for a further 3-4 h.After the incubation, the induced cells were centrifuged at 5 000 rpmand 4° C. (Heraeus fluorocentrifuge) for 15 min, and the pellet waswashed in PBS and then resuspended in 8 ml of 8 M urea lysis buffer. Thecells were disrupted by immersing the cells in liquid nitrogen and thenstoring at 37° C. 3 times. After the first nitrogen treatment, 0.75mg/ml lysozyme was added. The last incubation in liquid nitrogen wasfollowed by incubation at 16° C. for 20 min and then by short ultrasoundpulses each of 10 sec while cooling in an ice-water bath until thesolution had a water-like viscosity. After centrifugation at 13 000 rpmand 4° C. (Beckman J2-21; JS 13.1-Rotor) for 10 minutes, an induced 150μl sample was removed and mixed with 50 μL of 4×RotiLoad buffer (Roth),and the induction of the particular HC110-R protein fragment togetherwith the uninduced sample was checked by SDS-PAGE with subsequentCoomassie staining or Western blot analysis with a mouse anti-Hisantibody.

The remaining supernatant was transferred into a fresh vessel and storedat −20° C. for further purification by affinity chromatography.

13. Protein Purification by Metal Affinity Chromatography

The enrichment took place under denaturing conditions via the N-terminal6×His tag using the IMAC systems (‘Immobilized Metal AffinityChromatography’) on TALONspin columns from Clontech [Palo Alto, U.S.A.].The resin in the column was first separately equilibrated with 5 volumesof 8 M urea lysis buffer, pH 8.0, sedimented at 3 000 rpm and 4° C. for4 min and incubated together with the HC110-R protein supernatant at RTwith gentle shaking for 20 min. After centrifugation again, the resinwas washed 3 times with 10 times the volume of 8 M urea lysis buffer, pH8.0, with gentle shaking at RT for 10 min, and again centrifuged. Afterthe last washing step, the pellet was taken up in 1 ml of 8 M urea lysisbuffer and used to load the column; the latter was subsequently washedtwice with 3 times the volume of 8 M urea lysis buffer before elutionwith imidazole-containing 8 M urea lysis buffer in several fractionseach of 150 μl. The content of fusion protein in the fractions ismeasured by gel analysis and Bradford protein determination.

14. Protein Purification by Affinity Chromatography

Protein purification by metal affinity chromatography is to be describedby way of example here. The enrichment took place under denaturingconditions via the N-terminal 6×His tag using the IMAC systems(‘Immobilized Metal Affinity Chromatography’) on TALONspin columns fromClontech [Palo Alto, U.S.A.]. The resin in the column was firstseparately equilibrated with 5 volumes of 8 M urea lysis buffer, pH 8.0,sedimented at 3 000 rpm and 4° C. for 4 min and incubated together withthe HC-1 protein supernatant at RT with gentle shaking for 20 min. Aftercentrifugation again, the resin was washed 3 times with 10 times thevolume of 8 M urea lysis buffer, pH 8.0, with gentle shaking at RT for10 min, and again centrifuged. After the last washing step, the pelletwas taken up in 1 ml of 8 M urea lysis buffer and used to load thecolumn; the latter was subsequently washed twice with 3 times the volumeof 8 M urea lysis buffer before elution with imidazole-containing 8 Murea lysis buffer in several fractions each of 150 μl. The content offusion protein in the fractions is measured by gel analysis and Bradfordprotein determination.

15. Amino Acid Sequence Analysis

To check the deduced amino acid sequence starting from the cDNAfull-length clone HC110-R, the 3′ end consisting of 688 bp (Pos.2486-3182) with a preceding start codon and Kozak sequence was clonedinto the pRSET B-expression vector, and expression of the 21 kD protein(189 AA) in competent BL21(DE3)pLysS E. coli cells was induced by adding1 mM IPTG. The fusion protein provided with an N-terminal His tag wasenriched on a Talon matrix and concentrated and desalted by means ofCentricon tubes. Because of the N-terminal His tag, a partial C-terminalprotein sequencing of 1 nM of the 21 kD HC110-R protein was carried outby stepwise Schlack-Kumpf degradation (Schlack et al. 1926) in amodification of Boyd (Boyd et al. 1992) by TopLab (Martinsried).Sequencing of the eliminated amino acids took place in a PROCISE 492amino acid sequence (PE Applied Biosystems, Weiterstadt) and wasanalyzed using the PROCISE C reversed phase HPLC system consisting of anABI 140 C microgradient system and an ABI 785A UV/VIS detector andidentified using the PROCISE C control software and the ABI dataanalysis software.

25 pmol of the 21 kD HC110-R protein were cleaved internally with 2%trypsin (Roche Molecular Biochemicals, Mannheim) at 37° C. overnight. Atotal of four peptide fragments was selected by comparison of theHC110-R standard chromatogram after trypsin digestion with thechromatogram of the trypsin blank digestion in order to precludesequencing of tryptic autolysis products. These four peptide fragmentswere subjected to partial N-terminal protein sequencing by the method ofstepwise Edman degradation in a modification by Hunkapiller et al.(1983) by TopLab (Martinsried). The cleaved peptide fragments wereseparated and fractionated by HPLC. The peptide fraction blotted ontoImmobilon was introduced into the reaction chamber of the PROCISE 492amino acid sequencer (Applied Biosystems, Weiterstadt) and the aminoacids were separated in a 140 C-PTH-analyzer and UV detector 785 A(Applied Biosystems, Weiterstadt). The amino acids were quantitated byreversed phase HPLC and identified by comparison of retention times witha standard chromatogram constructed before the sequence analysis.

16. Cell Culture Lines

The following cell culture lines were purchased from the DeutscheSammlung von Microorganismen und Zellkulturen GmbH [Braunschweig][Drexler et al. 1995]:

-   -   COS-7 cells (DSM: ACC 60)—monkey, kidney    -   HEK-293 cells (ATCC: CRL 1573)—human, embryonic, kidney        17. Cultivation of the Various Cell Culture Lines

The adherent cell lines COS-7 and HEK-293 were cultivated in 110 mmtissue culture dishes [Greiner, Solingen] in a volume of 10 ml of mediumat 37° C., 5% CO₂ and 95% humidity. The cell culture was maintained bycultivating COS-7- and HEK-293 cells in DMEM medium. The media contained3.024 g/l NaHCO₃, 10% FCS, 50 U/ml penicillin and 50 μg/ml streptomycinand were heated to 37° C. before use. For subcultivation of the COS-7cells, they were stored at 4° C. for 2 h and then detached mechanicallyfrom the culture dish using a cell scraper. The HEK-293 cells could bedetached directly from the bottom of the culture dish using a glasspipette.

18. Transient and Stable Transfection of Eukaryotic Cells

The non-liposomal transfection reagent FuGENE 6 from Roche MolecularBiochemicals (Mannheim) was employed for transient introduction offoreign DNA into mammalian cells (Kurachi et al. 1998). For thispurpose, about 0.5-1.5×10⁵ cells in 2 ml of medium were put on 35 mmtissue culture dishes in which a sterile glass slide coated with 1%gelatin was placed for subsequent confocal laser scanning microscopy.The cells were cultivated at 37° C., 5% CO₂ and 95% humidity overnight.The medium was changed again before the transfection. For thetransfection, 3 μl of FuGENE 6 [Roche Molecular Biochemicals] werediluted in 97 μl of serum-free medium and incubated at RT for 5 min foreach reaction mixture. 100 μl portions of the diluted FuGENE 6 werepipetted dropwise onto 1-2 μg of plasmid-DNA (0.5-1 μg/μl) for eachmixture and, after cautious mixing, incubated at RT for a further 15min. The complete reaction mixture was then added dropwise to the cells,and the transfection mixture was distributed uniformly by gentlyswirling the dish. The cells were cultivated further for 1-2 dayswithout changing the medium. The following 3 negative controls werealways included with each transfection: a 35 mm tissue culture dish with0.5-1.5×10⁵ cells was not transfected, DNA, but no FuGENE 6, was addedto the transfection mixture in the second dish, and FuGENE 6, but noDNA, was added to a third dish.

For stable transfection, the desired plasmid HC110-R was cloned in thecorrect reading frame into the slightly modified expression vectorpSecTag A [Invitrogen, Leek, NL] or pIRESneo [Clontech, Palo Alto,U.S.A.]. These vectors harbour a resistance gene as marker so that onaddition respectively of Zeocin and G418 or bleomycin 48 72 h after thetransient transfection and at every subsequent change of medium onlysuccessfully transfected cells which permanently express the resistancegene product survive. The optimal concentration of Zeocin- orG418-containing selection medium was determined beforehand by setting upserial dilutions of the respective cell line.

19. Cellular Localization of Recombinant Proteins in Mammalian Cells

The complete coding region of the HC110-R DNA was cloned into theHindIII/SalI sitee of pEGFP-N3 in order to link the HC110-R proteinC-terminally to GFP (green fluorescent protein). The 137 kDa fusionprotein is expressed in transiently transformed recipient cell lines,which can be demonstrated by Western blot analysis. The CLSM (confocallaser scanning microscopy) shows that the HC110-R-GFP fusion protein islocalized in the cytoplasm of COS-7 and HEK-293 cells and, to a smallerextent, also on the plasma membrane (see also FIG. 8).

A Zeiss IM 35 microscope (Zeiss, Oberkochen) with a Leica CLSMattachment TCS NT (‘Confocal Laser Scanning Microscope Unit’, LeicaLasertechnik, Heidelberg), version 1.5.451, was used for the confocallaser scanning microscopy. Fluorescence of the GFP protein and of thedye fluorescein isothiocyanate (FITC) was excited at 488 nm with anargon laser and that of rhodamine, Texas red, phycoerythrin, Alexa 568,LysoTracker™ Red DND-99, MitoTracker™ Red CMX Ros and propidium iodidewas excited at 568 nm with a krypton laser. Z-Series of optical sectionsthrough the cell were scanned with a resolution of 1024×1024 pixels anda thickness of 0.5 μm (Giese et al. 1995). Evaluation took place withthe AVS software (Advanced Visual Systems Inc., Waltham, Mass., U.S.A.)and later with Adobe Photoshop 5.0 and Corel Draw 8.0 for Windows.

The fusion protein is located in particular in acidic lysosomes, asshown by colocalization using Lysotracker™, a probe for labelling acidicorganelles. The vesicles containing the HC110-R protein were observed inincreased numbers near the nucleus. A fusion protein of GFP and themouse β₂-adrenergic GPCR was prepared as control (Accession NumberX155643, Nakada et al. 1989), and transfection thereof resulted in thesame distribution pattern within the cell as in the case of theHC110-R-GFP fusion protein (FIG. 8).

20. EGFP Constructs for Transient Expression

The vectors pEGFP C1 and pEGFP N3 (Clontech, Palo Alto, Calif., U.S.A.)were cut with the restriction enzymes Hind III and Sal I. Amplificationof the HC110-R full-length cDNA took place with the P83EGFP_ATG HindIII5′ primer 5′-GGT AGA AGC TTT TCG GTT TAA TAC CAA CAT GAG G-3′ and theP3057EGFP_o.TGA SalI 3′ primer 5′-CTG TGT CGA CAA CAT TTC GCC AAT AGTTAG G-3′ at an annealing temperature of 65° C. The PCR product was thenlikewise cut with the enzymes Hind III and SalI, and was ligated togenerate an open reading frame between the Hind III and SalI cleavagesites of the particular vector and was transformed. The resulting fusionprotein with the N-terminal GFP tag was called GFP-HC110-R and that withthe C-terminal EGFP tag was called HC110-R-GFP. A mouse O₂ adrenergicreceptor (GenBank Accession No.: P18762; Nakada et al. 1989) was fusedwith a C-terminal EGFP tag by amplifying the full-length cDNA with theP117 mouse 2AR XhoI 5′ primer 5′-TAC CTC GAG CTG CTA ACC TGC CAG CCATG-3′ and the P1349 mouse β₂AR EcoRI 3′ primer 5′-TGT AGA ATT CTT CCTTCC TTG GGA GTC AAC GCT-3′ using an annealing temperature of 55/60° C.,cutting with the restriction enzymes XhoI and EcoRI and ligating intothe XhoI-EcoRI linearized pEGFP N3. The full-length cDNA of a humanmuscarinic receptor 1 (huM1Rez.; GenBank Accession No.: Y00508; Allardet al. 1987) which had previously been amplified with the P70HumM1RezXhoI 5′ primer 5′-ATA TCT CGA GAG CCC CAC CTA GCC ACC ATG AAC A-3′ andthe P1465HumM1Rez EcoRI 3′ primer 5′-GAC GAA TTC CAT TGG CGG GAG GGA GTGCGG T-3′ at 55/60° C. was likewise ligated into the XhoI-EcoRI-cut pEGFPN3 vector. The resulting fusion proteins with an open reading frame werecalled mouseβ₂AR-EGFP and huM1Rez-EGFP.

21. HC110-R-MycHis Tag Constructs for Stable or Transient Transfection

The vector pMyc6×His is derived from the vector pSecTagA [Invitrogen,Leek, NL] by double digestion with the restriction enzymes NhiI andSfiI, followed by blunting of the ends and religation of the vector. Thecoding region of HCl 0-R was amplified using the PCR primersP83MycTag_ATG BamHI 5′ primer 5′-ATA GGA TCC TTC GGT TTA ATA CCA ACA TGAGG-3′ and P3058MycTag_o.TGA XbaI 3′ primer 5′-CCT GTC TAG AAA CAT TTCGCC AAT AGT TAG G-3′ at an annealing temperature of 56/60° C., cut withthe enzymes BamHI and XbaI, and ligated into the pMyc6×His vector whichhad likewise been linearized with BamHi XbaI and transformed into E.coli DH5α cells. Subsequently, COS-7 cells were stably transfected withthe construct and maintained under Zeocin selection pressure.

In parallel with this, the HC110-RMycHis cDNA was amplified with theprimers P83_ATGNotI-5′ 5′-ATA TTG CGG CCG CTT CGG TTT AAT ACC AAC ATG-3′and pMycHis_TGABamHI-3′ 5′-CGC GGA TCC TAG AAG GCA. CAG TCG AGG-3′, thencut and ligated into the bicistronic expression vector pIRES1neo(GenBank Accession No.: U89673) (Clontech, Palo Alto, U.S.A.) which hadlikewise been restricted with BamHI and NotI. The pIRES1neo vectoradditionally contains an internal ribosome binding site (IRES) of theencephalomyocarditis virus (ECMV) shortly before the start ATG of theneomycin resistance gene (Rees et al. 1996). In this way, two openreading frames, that of HC110-R and that of the antibiotic resistancemarker, are translated from only one mRNA with a human cytomegalovirus(CMV) promoter (Jackson et al. 1990; Jang et al. 1988). Selection tookplace with G418 (Calbiochem-Novabiochem, La Jolla, Calif., U.S.A.) inCOS-7 and HEK-293 cells.

22. Confocal Laser Scanning Microscopy

A Zeiss IM 35 microscope (Zeiss, Oberkochen) with a Leica CLSMattachment TCS NT (‘Confocal Laser Scanning Microscope Unit’, LeicaLasertechnik, Heidelberg), version 1.5.451, was used for the confocallaser scanning microscopy.

Fluorescence was excited at 488 nm with an argon laser and at 568 nmwith a krypton laser. Z-Series of optical sections through the cell werescanned with a resolution of 1024×1024 pixels and a thickness of 0.5 μm(Giese et al. 1995). Evaluation took place with the AVS software(Advanced Visual Systems Inc., Waltham, Mass., U.S.A.) and later withAdobe Photoshop 5.0 and Corel Draw 8.0 for Windows.

23. Studies of Binding of α-LTX to HC110-R Fragments

Proteins (20 μg/lane) were fractionated by SDS-PAGE (Lämmli et al.,1970) and electroblotted onto nitrocellulose membranes. In the case ofα-LTX binding experiments, the blots were incubated with α-LTX at aconcentration of 20 nM in TST at room temperature for 2 h. After washingwith TST three times, an anti-α-LTX antibody (from rabbits,Calbiochem-Novabiochem) was used at a concentration of 0.1 μg/ml and anantibody conjugated with peroxidase and directed against rabbit IgG(from goat, dilution: 1:25 000) was used. The antibodies were detectedin all experiments using ECL (Amersham-Pharmacia). See also FIG. 9.

24. Calcium Imaging

The intracellular free Ca²⁺ concentration [Ca²⁺]_(i) was measured inCOS-7 and HEK293 cells transiently transfected with the HC110-R-EGFP orβ₂-AR-EGFP construct using the calcium imaging method (see also FIG.11). In each case 2×10⁵ COS-7 and HEK293 cells were applied to a 42 mmcover glass coated with 1% gelatine in 55 mm tissue culture dishes with5 ml of DMEM medium (with 10% FCS and pen/strep). The transfection tookplace with the aid of the non-liposomal transfection reagent FuGENE 6(Roche Molecular Biochemicals, Mannheim). This entailed 6 μl of FuGENE 6being incubated in 200 μl of DMEM medium without FCS initially at RT for5 min and, after addition of 4 μg of plasmid DNA, at RT for a further 15min, before the complete transfection mixture was pipetted dropwise ontothe cells. Constructs of the HC110-R full-length clone with a GFP tagfused in the correct reading frame, and the complete cDNA of theβ₂-adrenergic receptor which had likewise been provided with a GFP tagat the 3′ end, were transfected. 4 μg of plasmid DNA of the purepEGFP-N3 expression vector (Clontech, Heidelberg) and untransfectedcells to which the transfection reagent FuGENE 6 had been added withoutDNA served as controls.

Loading of the cells with 1 μM Fura-2/acetoxymethyl ester (Fura-2/AM)(Sigma, Deisenhofen) took place 48 h after the transfection in anNa⁺-HBS solution (150 mM NaCl; 5.4 mM KCl; 1.8 mM CaCl₂; 0.8 mM MgSO₄7H₂O; 20 mM glucose; 20 mM Hepes in H₂O, pH 7.3) at 37° C., 5% CO₂ and95% humidity for 30 min. After the incubation, the cells loaded withFura-2/AM were examined under an inverted microscope (Zeiss, Axiovert,Germany) and, in parallel with this, in a digital imaging fluorescencemicroscope (PTI) and differentiated into transfected and untransfectedcells at a wavelength of 440 and 490 nm respectively. On average, 5-7transfected and non transfected cells were selected by fixing an ROI(region of interest). The extinction was measured at 340 nm(calcium-bound Fura-2/AM) and 380 nm (free Fura-2/AM), and the emissionwas measured at 510 nm. Evaluation took place using the Image Master 1.xsoftware by forming the quotient of 340:510 nm, 380:510 nm and the ratioof 340/380:510 nm (background corrected images) as a function of theagent added.

Agents were always added 6 min after starting the measurement byremoving an appropriate volume of Na⁺ HBS buffer and pipetting the agentdirectly into the sample vessel for a further 30 to 50 min. The totalvolume in the sample vessel was constant at 1.5 ml throughout theexperiment. Initially 30 nM and 75 nM α-latrotoxin (α-LTX, RBI. Natick,U.S.A.) were added to the COS-7 cells expressing the HC110-R-GFP fusionprotein, and various concentrations of α-LTX (7.5 nM; 25 nM; 50 nM, 75nM, 90 nM and 120 nM), to determine the dose-dependency, and variousconcentrations of the cyclic depsipeptide BAY 44-4400 (100 ng/ml (89.3nM), 333 ng/ml (297 nM); 400 ng/ml (357 nM), 1 μg/ml (893 nM), 10 μg/ml(8.9 μM), to determine the optimal active concentration, were added tothe HEK-293 cells expressing HC110-R-GFP, in each case 6 min beforestarting the experiment. In some experiments, the HEK-293 cells werepreincubated with 4 or 400 ng/ml BAY 44-4400, or with 4 or 400 ng/ml ofthe optical antipode PF1022-001, which has no anthelmintic activity, inNa⁺ HBS at 37° C., 5% CO₂ and 97% humidity for 90 min, and the cellswere loaded after 60 mm by adding 1 μM Fura-2/AM to the BAY 44-4400solution or to the PF1022-001, for the remaining 30 min. After the cellshad been introduced into the apparatus, 75 nM α-LTX were also addedthereto 6 min after starting the experiment. The optimal α-LTXconcentration of 75 nM was tested on HEK-293 cells which express theβ₂-adrenergic receptor with C-terminal GFP tag (β₂-R-GFP). CdCl₂ (ineach case 1 μM and 10 μM) was dissolved in Na⁺ HBS, and nifedipine (15μM) and EGTA (2 mM) were dissolved in 0.1% DMSO. Addition of CdCl₂ andnifedipine took place immediately at the start of the experiment andbefore addition of α-LTX, and EGTA was added both at the start of theexperiment and 4 min after the addition of α-LTX. Dissolved α-LTX waspresent in a concentration of 300 nM in 50 mM tris-HCl, pH 8.0; BAY44-4400 was initially dissolved in pure DMSO (stock solutions between0.004 and 10 μg/ml). The stock solutions were adjusted to the requiredconcentration in the Na⁺ HBS buffer. The maximum amount of the activesubstance BAY 44-4400 which could be dissolved at a final concentrationof 0.1% DMSO was found by setting up serial dilutions in Na⁺ HBS.Neither 0.1% DMSO nor any other test component interacted with Fura-2/AMat the chosen concentrations.

The data were analyzed using the Excel 98 table calculation programme.The results are derived from at least 2-4 reproduced experiments on ineach case 4-8 transfected and un-transfected cells.

The intracellular calcium concentration was calculated by the method ofGrynkiewicz et al. (1985) after previous calibration by the method ofMcCormack et al. (1991).

25. Binding of PF1022A to HC110-R

The binding of PF1022A and the morpholine derivative BAY44-4400 toHC110-R is to be shown by SDS-PAGE, ligand precipitation and analyticalflow cytometry below. It is additionally intended to check whetherPF1022A and its derivatives lead—like α-LTX—to changes in [Ca²⁺]_(i) inHC110-R-transfected HEK-293 cells or else affect the HC110-R-mediatedα-LTX signalling.

Occasionally, antibodies or proteins which give clear signals inimmunofluorescence show no reaction on use of the immunoblot method.Since denaturation of proteins is necessary for carrying out SDS-PAGE,there may be destruction of conformation-dependent epitopes. Antibodiesor proteins which specifically recognize such epitopes may therefore nolonger bind. Thus, HC110-R-specific binding in a Western blot is notidentifiable either with the biotinylated active substance PF1022A orwith the morpholine derivative BAY44-4400, which is more soluble thanPF1022A. BAY44-4400 differs from PF1022A by 2 morpholine residues whichare covalently bonded to the phenyl rings of the two D-phenyllactylradicals of PF1022A. However, if the proteins are renatured in the SDSseparating gel using urea, which brings about partial removal of the SDSthrough abolishing hydrophobic interactions, it is possible to detectHC110-R-specific binding in the Western blot. For this purpose, totalprotein from untransfected HEK-293 cells and HEK-293 cells whichtransiently express HC110-R-Myc/His, and from the 54 kDa N terminus andthe 21 kDa C terminus of HC110-R which were expressed in E. coli, werefractionated by SDS-PAGE, renatured and blotted in the usual way.Immunodetection took place after incubation with the ligand BAY444400,with a rabbit anti-PF1022A-KLH immune serum and with a goat anti-rabbitIgG HRP antibody. In addition, a partially renatured SDS polyacrylamidegel with the protein fractions of untransfected HEK-293 cells, HEK-293cells transiently transfected with HC110-R-Myc/His, and the N terminusof HC110-R expressed in E. coli and total protein from H. contortus wasblotted, incubated with PF1022A-biotin and detected using streptavidinHRP. Each of the two blots showed a distinct band 116 kDa in size in theHEK-293 cells which stably expressed HC110-R-Myc/His, whereas the laneswith untransfected cells showed no band at this level. In addition, boththe morpholine derivative BAY444400 and the biotinylated PF1022A boundto the 54 kDa N terminus of HC110-R, but the 21 kDa C terminus showed nospecific PF1022A binding. It was possible to detect with thebiotinylated PF1022A a total of 2 bands in the total protein fractionfrom adult H. contortus nematodes: a band 110 kDa in size and anotherband about 88 kDa in size. The latter is very probably a biotinylatedprotein like that of 83 kDa which has already been described for thenematode Heterakis spumosa, especially since, in contrast to the band110 kDa in size, it is detected even if detection takes placeexclusively with streptavidin HRP. On the other hand, HEK-293 cellsstably transfected with HC110-R-Myc/His no longer show a band 116 kDa insize with streptavidin HRP alone. It was furthermore possible topreclude nonspecific signals being caused by the goat anti-rabbit IgGHRP secondary antibody alone.

In order to verify these results further, the following ligandprecipitation was carried out. 2 mg and 4 mg portions of magnetic DynalM-280 streptavidin-coupled beads were mixed with 500 μg of biotinylatedPF1022A. In a control mixture with 4 mg of Dynal beads, TST buffer wasadded in place of PF1022A. Excess PF1022A-biotin was removed by magneticseparation before a mixture of the HC110-R terminus and C terminusexpressed in E. coli and previously purified by affinity chromatographywas added. After renewed magnetic separation to remove unbound HC110-Rfragments, one aliquot derived from 2 mg, and one from 4 mg, of Dynalbeads was fractionated by SDS-PAGE and blotted. Detection of theprecipitate took place via the N-terminally fused His tag with a mouseanti-His IgG and a rabbit anti-mouse IgG HRP antibody. Only theN-terminal 54 kDa region of HC110-R is precipitated by PF1022A, andaccordingly the C terminus of HC110-R, which is not bound to PF1022A, ispreviously removed by magnetic separation. Nonspecific binding of the Nor C terminus of HC110-R to the streptavidin-coupled beads was precludedby adding no biotinylated PF1022A to a reaction mixture.

Binding of the ligand PF1022A in vivo to HC110-R was additionallyexamined by FACS analysis (FIG. 13). For this purpose, HEK-293 cellswere transiently transfected with HC110-R-GFP or GFP-HC110-R or elsetransiently cotransfected with HC110-R-Myc/His and GFP. UntransfectedHEK-293 cells, and HEK-293 transiently transfected with β₂-R-GFP orM1-R-GFP were employed as controls. 24 h after the transfection, thecells were incubated with biotinylated PF1022A, and PF1022A-bound cellswere detected using streptavidin-phycoerythrin and were finally fixed. Anegative control of cells transfected with HC110-R-GFP was incubatedonly with streptavidin-phycoerythrin without PF1022A-biotin.

The FACScan was used with an excitation wavelength of 488 nm for thefluorescence analysis of in each case 10000 HEK-293 cells for their cellsize, granularity and fluorescence staining. Phycoerythrin has—likeTRITC—an absorption spectrum which overlaps with EGFP, while theemission spectra are adequately separated, so that both fluorochromescan be excited at only one wavelength. Cell detritus was excluded fromthe measurement of the fluorescence intensity by setting a ‘gate’ on themain cell population. For quantitative evaluation, the limits were fixedfor negative, unstained and positive, GFP-fluorescent cells on the basisof the untransfected and GFP-transfected cells. The 4.6% ofgreen-fluorescent, untransfected HEK-293 cells (autofluorescence) weresubtracted from the other GFP-fluorescent samples; and the value for thenonspecific staining of the cells by the phycoerythrin-coupledstreptavidin (5.4%) was subtracted from all red-fluorescent cells (Tab.1). TABLE 1 Detection of PF1022A binding to HEK-293 cells transfectedwith HC110-R by FACScan Phycoerythrin Plasmid GFP fluorescencefluorescence Untransfected  0 ± 0% 0 ± 0% GFP 17.0 ± 0.5% 1.3 ± 0.4%HC110-R-GFP 10.9 ± 0.2% 4.5 ± 0.4% GFP-HC110-R  9.4 ± 2.5% 2.5 ± 0.3%β₂-R-GFP  7.6 ± 0.4% 0.8 ± 0.1% M1-R-GFP  8.2 ± 0.7% 1.6 ± 0.1%HC110-R-Myc/His + GFP 23.1 ± 2.3% 10.3 ± 0.7% 

Calculation of the percentage of green-fluorescent cells which were alsored-fluorescent after subtraction of the autofluorescence and thenonspecific red staining by streptavidin-phycoerythrin showed that 41.2%of the cells expressing HC110-R-GFP, 26.8% of the cells expressingGFP-HC110-R and 44.5% of the cells cotransfected with HC110-R-Myc/Hisand GFP bind PF1022A, whereas only 7.5% of the cells expressing GFP,9.9% of the cells expressing 2-R-GFP and 19.2% of the cells expressingM1-R-GFP bind PF 1022A (FIG. 13).

26. Interactions of PF1022A Derivatives with HC110-R-mediated α-LTXSignal Transduction

PF1022A shows in vitro neuropharmacological activity between 10-9 and10-3 mg/ml depending on the nematode species. In order to detect anyinterference of PF1022A and its derivatives with HC1110-R and the α-LTXsignalling mediated by HC110-R, the Ca imaging method was used toinvestigate the effect of BAY44-4400, a more soluble morpholine variantof PF1022A, on HEK-293 cells transiently or stably expressing HC110-R.The optical antipode to PF1022A, PF1022-001, which has an activity whichis more than 100 times less in vitro and in vivo was employed ascontrol. BAY44-4400 and PF1022-001 were initially dissolved in pure DMSObecause of their hydrophobicity. The stock concentration was chosen sothat there was always only 0.1% DMSO present in the experimentalmixture. It was possible in control experiments to preclude DMSOconcentrations up to and including 0.1% leading to an impairment of theexperimental results in untransfected HEK-293 cells and HEK-293 cellstransfected with HC110-R-GFP (FIG. 14A). Fura-2-loaded untransfectedcells and cells transfected with HC110-R-GFP were stimulated on the onehand with 100, 300 or 400 ng/ml and 1 or 10 μg/ml BAY44-4400 and on theother hand with the same concentrations of PF1022-001. At noconcentration was a Ca²⁺-mediated response detectable over the entireperiod of 50 min. FIGS. 14B and C show by way of example the stimulationof cells transfected with HC110-R-GFP and M1-R-GFP with 400 ng/mlBAY444400 and PF1022-001. Since the PF1022A derivatives are possiblyalso taken up by the cells and thus may intervene secondarily in signaltransduction pathways, and because the PF1022A derivatives remain for aprolonged period in the worm, depending on the parasite, the cells werepreincubated with the derivatives for 90 min in some experiments. Cellspreincubated with 4 ng/ml or 400 ng/ml BAY44-4400 or PF1022-001 for 90min showed no change in [Ca²⁺]_(i) (FIG. 14D). On direct addition ofconcentrations of 300 ng/ml BAY44-4400 and above, it was possible—incontrast to the optical antipode—to observe on a monitor whichrepresented the cells with 40× magnification a slight swelling of thecells which was, however, reversible within a few minutes, as directeffect of the anthelmintic in HEK-293 cells transfected withHC110-R-GFP.

Very recent experiments have shown that piperazin synergisticallyenhances the effect of PF1022A/BAY44-4400 in experiments with Heterakisspumosa in vitro and in vivo. When 400 ng/ml BAY44-4400 was givensimultaneously with 1 or 10 μM piperazine (FIG. 14F), no change in[Ca²⁺]_(i) was detectable either in untransfected HEK-293 cells or inHEK-293 cells transiently transfected with HC110-R-GFP. The controlmixture with 10 μM piperazine likewise led to no change in the Ca²⁺balance in untransfected and HC110-R-GFP-transfected cells (FIG. 14E).

In contrast to this, both BAY44-4400 and the optical antipode PF1022-001influenced, in the presence of 75 nM α-LTX, the α-LTX-induced signaltransduction of HC110-R-GFP-expressing HEK-293 cells, although theextent varied (FIGS. 14B-E). In a control experiment in which cellstransfected with HC110-R-GFP or M1-R-GFP were treated with 75 nM α-LTX,0.1% DMSO, 6 min before the stimulation, it was possible to preclude aneffect on the α-LTX-induced change in [Ca²⁺]_(i) exerted by the solventused for BAY444400 and PF1022-001 at the latter concentration.HC110-R-GFP-expressing cells showed, in contrast to theM1-R-GFP-expressing cells, the previously described biphasic profilewith comparable values of [Ca²⁺]_(i) after addition of α-LTX (FIG. 15A).Incubation of HC110-R-GFP-expressing cells with 4 ng/ml BAY44-4400 6 minbefore addition of α-LTX diminished the described effect of α-LTX on theCa²⁺ concentration: the first small increase in [Ca²⁺]_(i) induced byα-LTX disappeared and the second delayed peak was reduced to 44±6.0 nMCa²+14 min after the addition of α-LTX (FIG. 15B). In the presence of 4ng/ml PF1022-001—the addition took place 6 min before stimulation withα-LTX—there was a rapid increase of 103±11.5 nM Ca²⁺, (FIG. 15B). Onaddition of 400 ng/ml BAY44-4400 to HC110-R-GFP-expressing cells 6 minbefore the addition of α-LTX there was—like with 4 ng/ml BAY44-4400(FIG. 15B)—a delayed increase in [Ca²⁺]_(i) by 49±4.2 nM after 23 min,which fell 8 min later to a plateau which was slightly elevated by11±1.4 nM compared with the initial values (FIG. 15C). On addition of400 ng/ml PF1022-001 6 min before incubation with 75 nM α-LTX there wasa rapid increase in Ca²⁺, of 112+14.3 nM Ca²⁺ (FIG. 15C), as previouslyobserved on addition of 4 ng/ml PF1022-001 (FIG. 15B). In another designof experiment, HC110-R-GFP-expressing HEK-293 cells were in each casepreincubated with 4 ng/ml (FIG. 15D) or 400 ng/ml BAY44-4400 orPF1022-001 (FIG. 15E) for 90 min. Active substance which had not beenbound or taken up was carefully washed out after the incubation andloading of the cells with Fura-2/AM before the cells were stimulatedwith 75 nM α-LTX. HC110-R-transfected cells preincubated with theoptical antipode PF1022-001 showed an increase in [Ca²⁺]_(i) of 102±6.4nM (FIG. 15D) with 4 ng/ml PF1022-001 and a comparable increase of109±8.4 nM Ca²⁺ (FIG. 15E) with 400 ng/ml. This increase in Ca²⁺, whichwas observed only a few minutes after addition of α-LTX, resembled therapid changes in [Ca²⁺]_(i) previously observed in FIGS. 15B and C whenPF1022-001 was added immediately before stimulation with α-LTX. Bycontrast, BAY44-4400 showed different responses in relation to theα-LTX-induced Ca²⁺ influx: on preincubation of HC110-R-GFP-expressingcells with 4 ng/ml BAY44-4400 for 90 min, α-LTX induction led to amaximum increase in [Ca²⁺]_(i) of 95±20.5 nM at 10 min before it fell toan extensive plateau elevated by 23±2.6 nM Ca²⁺ (FIG. 15D). Onpreincubation with 400 ng/ml BAY44-4400, α-LTX stimulation was followedby an increase in [Ca²⁺]_(i) by a maximum of 65+7.5 nM (FIG. 15D), whichwas moreover delayed by 12 min compared with preincubation with 4 ng/mlBAY44-4400 (FIG. 15C).

On addition of 400 ng/ml BAY44-4400 to HEK-293 cells stably expressingHC110-R-Myc/His 6 min before addition of α-LTX there is completeinhibition of the observed immediate increase in [Ca²⁺]_(i) by 267+45.8nM (FIG. 15F). In contrast to this, addition of 400 ng/ml PF1022-001leads to no significant change in [Ca²⁺]_(i), the increase in Ca⁺ being209±33.4 nM Ca (FIG. 15F).

In order to show the specific effect of BAY44-4400 on the α-LTX-mediatedCa²⁺ signalling in HC110-R-expressing cells, the following controlexperiments were carried out: initially untransfected HEK-293 cells werestimulated with 1 mM carbamylcholine chloride (carbachol), a ligand formuscarinic acetylcholine receptors, in the presence and absence of 400ng/ml BAY44-4400 (FIG. 15G). In both cases there was a comparableimmediate increase in [Ca²⁺]_(i) by endogenous, natural muscarinicacetylcholine receptors by 58±8.1 nM Ca²⁺ for HEK-293 cells in thepresence of BAY44-4400 and by 53+6.8 nM Ca²⁺ in the absence of theanthelmintic. Additional transient transfection of HEK-293 cells withthe human muscarinic M1 acetylcholine receptor C-terminally fused to GFP(M1-R-GFP) led to a slight increase in [Ca 2+], compared withuntransfected cells (FIG. 15G), by 89±5.5 nM in the presence and by85±6.1 nM Ca²⁺ in the absence of 400 ng/ml BAY44-4400 (FIG. 15H). Norwas any significant effect of BAY44-4400 on M1-R-GPCR observable.

Stimulation of the endogenously present natural β₂-adrenergic receptorsin untransfected HEK-293 cells with 1 mM isoproterenol, or the nicotinicreceptors with 1 mM arecoline—as previously described for carbachol—inthe presence and absence of 400 ng/ml BAY44-4400 once again led to nosignficant difference in [Ca²⁺]_(i) (FIG. 15I). Isoproterenol induced anincrease of 45±4.7 nM in [Ca²⁺]_(i) in the presence and an increase of48±5.7 nM Ca²⁺ in the absence of BAY44-4400. Following arecolinestimulation there was an increase of 28±4.1 nM in Ca²⁺ in the presenceof BAY44-4400 and of 27±3.5 nM in Ca²⁺ in the absence of the activesubstance (FIG. 151).

27. Obtaining Antibodies

Antibodies were obtained by employing female NMRI mice and rabbits(chinchilla crosses). For immunization of the NMRI mice, 3×15 μg of thepurified 21 kD C-terminal HC110-R protein fragment were each dissolvedin 100 μl of PBS⁻ together with 100 μl of FCA and injectedsubcutaneously into two naïve NMRI mice on days 1, 8 and 15. Bloodsampling and obtaining of serum took place on day 23.

To obtain serum, the blood obtained by cardiac puncture was incubatedinitially at 37° C. for 1 h and then at 4° C. for at least 2 h. Thecellular constituents were subsequently pelleted twice at 3 000 rpm and4° C. in the Beckman GPKR centrifuge for 10 min, and the supernatant wasinactivated at 56° C. for 45 min. After a further centrifugation at 13000 rpm and 4° C. in the Heraeus biofuge 15R for 10 minutes it waspossible to take off the serum and store it at −20° C. until usedfurther.

After the first blood sampling and obtaining of preimmune serum, tworabbits initially received subcutaneous injections of 50 μg of theC-terminal 21 kD HC110-R protein and of the 54 kD N-terminal HC110-Rprotein in a suspension consisting of equal parts of PBS⁻ and FCA. Twofurther immunizations each with 100 μg of antigen took place on day 31and day 79 after the first immunization. A first blood sampling wascarried out on day 43. A second blood sampling and obtaining of serumtook place on day 98 after the first immunization.

DESCRIPTION OF THE FIGURES

FIG. 1: HC110-R mRNA and protein

-   (A) Northern blot analysis using total RNA from Haemonchus contortus    and the 3.6 kbp cDNA of HC110-R.-   (B) 10% SDS-PAGE fluorogram of in vitro translated ³⁵S-labelled    HC110-R protein. In vitro translation of 1 μg of in vitro    transcribed HC110-R mRNA (HC110-R), negative control without HC110-R    mRNA (control), 1 μg of luciferase mRNA as positive control    (luciferase).

FIG. 2: Full-length cDNA sequence of HC110-R and derived amino acidsequence

The Kozak motif (Kozak, 1989) and the polyadenylation signal of HC110-R(GenBank Accession No.: AJ272270) are underlined; the start codon inposition 100 is emboldened. Signal peptide (residues 1-21, bold),lectin-like sequence (residues 22-125, dotted), Thr stretch (residues128-147, grey background); cysteine-rich region of structureCX₉WX₁₂CX₉WXCX₅WX₉CX₃W (residues 166-221, wavy line, Cys and Trpresidues additionally bold); 4-Cys region of structure CXWWX₆WX₄CX₁₁CXC(residues 478-524, broken line, Cys and Trp residues additionally bold);the 7 transmembrane regions (between residues 563-772, bold andunderlined); the proline-rich stretch (residues 845-861, greybackground), the PEST region (residues 915-933, grey background); theN-glycosylation sites (residues 26, 499 and 862, bold); the highlyconserved putative disulphide linkage of the Cys-Cys pair betweensecretin GPCRs (position 595 and 666, bold and double underlining).

FIG. 3: Alignment of the derived amino acid sequences of the H.contortus HC110-R and Caenorhabditis elegans cosmid clone B0457(CE-B0457; GenBank Accession No.: Z54306)

Signal peptide (SP, residues 1-21, bold), lectin-like sequence (lectin,residues 22-125, dotted); Thr stretch (T-rich, residues 128-147, greybackground); Cys-rich region of structure CX₉WX₁₂CX₉WXCX₅WX₉CX₃W (Csignature, residues 166-221, wavy line, Cys and Trp residuesadditionally bold); 4-Cys motif of structure CXWWX₆WX₄CX₁₁CXC (4 Cregion, residues 478-524, broken line, Cys and Trp residues additionallybold); the 7 transmembrane regions (between residues 536-772, bold andunderlined); the Pro-rich stretch (P-rich, residues 845-861, greybackground); the PEST region (PEST, residues 915-933, grey background);the N-glycosylation sites (residues 26, 499 and 862, bold); the highlyconserved putative disulphide linkage of the Cys-Cys pair betweensecretin GPCRs (position 595 and 666, bold and double underlining).Identical amino acids are marked with asterisks, very closely relatedamino acids with a colon, related amino acids with a single dot.

FIG. 4: Alignment of the 7 transmembrane receptor HC110-R and severalother receptors of the secretin subfamily

HC110-R: Haemonchus contortus heptahelical orphan transmembrane receptor(Accession no.: AJ272270); BTLAT3: Bos taurus latrophilin-3 (severalsplicing variants, Accession no.: G4164053-G4164075); RNLAT1: Rattusnorvegicus latrophilin-1 (Accession no.: U78105, U72487); MMEMR1: Musmusculus EMR1 hormone receptor precursor (Accession no.: Q61549);HSCD97: Homo sapiens leukocyte activation antigen CD97 (Accession no.:P48960); MMCADH: M. musculus cadherin 7 transmembrane receptor precursor(Accession no.: G3800738); XLXRF1: Xenopus laevis corticotropinreleasing receptor precursor (Accession no.: 042602); RNVIP1: R.norvegicus vasoactive intestinal polypeptide receptor precursor 2(Accession no.: Q02643). The seven transmembrane domains have a greybackground (I-VII) and the highly conserved putative disulphide linkageis emboldened.

FIG. 5: Alignment of the derived amino acid sequences of the H.contortus HC 110-R and R. norvegicus latrophilin-1 (GenBank Acc. No.U78105, U72487)

Signal peptide (SP, residues 1-21, bold), lectin-like sequence (lectin,residues 22-125, dotted); Thr stretch (T-rich, residues 128-147, greybackground); Cys-rich region of structure CX₉WX₁₂CX₉WXCX₅WX₉CX₃W (Csignature, residues 166-221, wavy line, Cys and Trp residuesadditionally bold); 4-Cys motif of structure CXWWX₆WX₄CX₁₁CXC (4 Cregion, residues 478-524, broken line, Cys and Trp residues additionallybold); the 7 transmembrane regions (between residues 536-772, bold andunderlined); the Pro-rich stretch (P-rich, residues 845-861, greybackground); the PEST region (PEST, residues 915-933, grey background);the N-glycosylation sites (residues 26, 499 and 862, bold); the highlyconserved putative disulphide linkage of the Cys-Cys pair betweensecretin GPCRs (position 595 and 666, bold and double underlining).Identical amino acids are indicated by asterisks, very closely relatedamino acids by a colon related amino acids by a single dot.

FIG. 6: Structure of the HC110-R proteins and of the rat latrophilin-1

-   (A) Hydrophobicity blot of HC110-R with the 7 transmembrane domains.-   (B) Protein structure of HC110-R with the following characteristic    motifs: signal sequence (SP), lectin domain (lectin), Thr stretch    (T-stretch), Cys motif (signature), 4-Cys region (4C region), 7    transmembrane domains (1-7, black), the Pro-rich motif (P-rich) and    the PEST sequence (PEST). The putative glycosylation sites are    depicted underneath the diagram (N), as are the Cys residues of the    4C region and the two conserved Cys residues of the putative    disulphide linkage (thin line without capital letters).-   (C) Protein structure of latrophilin-1. Latrophilin-1 has no Thr    stretch but contains an additional olfactomedin binding motif    (olfactomedin), a Pro-Thr region (P-T region), a linker domain    (linker) and a long region containing several repeats (long).

FIG. 7: Expression of HC110-R-GFP in HEK-293 and COS-7 cells

-   (A) Total protein from HEK-293 and COS-7 cells transiently    transfected with GFP (GFP) or HC110-R-GFP (HC110-R) and    untransfected (n. t.) HEK-293 and COS-7 cells was isolated by the    Trizol method 24 h after transfection.-   (B) Total protein from HEK-293 cells transiently transfected with    β₂-R-GFP (β2-R) or M1-R-GFP (M1-R), and untransfected (n. t.)    HEK-293 cells was isolated by the Trizol method 24 h after the    transfection. SDS marker bands at 116, 90, 70 and 55 kDa (marker).

20 μg of total protein per lane were fractionated on a 10% SDSpolyacrylamide gel and blotted onto a nitrocellulose membrane. Blockingin RotiBlock solution overnight was followed by immunodetection usingthe monoclonal mouse anti-GFP IgG primary antibody (0.4 μg/ml) and themonoclonal rabbit anti-mouse IgG-HRP secondary antibody (1:25000) by theECL system.

FIG. 8: Cellular localization of HC110-R and β₂-R in transfected COS-7cells COS-7 cells were transfected with HC110-R-GFP and β₂-R-GFP both ofwhich carry a C-terminal GFP tag. The CLSM was carried out 24 hoursafter the transfection. The bars correspond to 10 μm.

-   (A) HC110-R-GFP is expressed on the plasma membrane and in the    cytoplasmic compartments. A compaction of cytoplasmic vesicles can    be seen in the vicinity of the nucleus.-   (B) CLSM colocalization of the green HC110-R-GFP protein with acidic    lysosomes, stained with LysoTracker Red DND-99 at 37° C. for 1 h.-   (C) β₂-R-GFP-transfected cells show a similar GFP fluorescence    pattern to HC110-R-GFP, or as described under (A). The receptor can    be localized to the plasma membrane and in vesicles and can in some    cases be colocalized with acidic lysosomes, stained with LysoTracker    Red DND-99 at 37° C. for 1 h.-   (D) Expressed β₂-R-GFP colocalizes in some cases with the    endoplasmic reticulum, stained with monoclonal anti-KDEL antibodies.

FIG. 9: Binding of α-latrotoxin to HC110-R

(A) 5 μg of total protein from isolated Latrodectus revivensis venomglands (1), and pure 130 kDa (X-LTX (2), also in each case 40 μg oftotal protein from HEK-293 cells stably transfected with theHC110-R-Myc/His construct in the pIRES1neo vector (3), untransfectedcells (4) and the 54 kDa N terminus (6) and 21 kDa C terminus (7) ofHC110-R induced in E. coli were fractionated in a 10% SDS polyacrylamidegel, blotted and blocked overnight. The denatured proteins wereincubated with 20 nM α-LTX for 2 h and possible bindings of α-LTXdetected with a rabbit anti-α-LTX IgG-HRP (1:5000), a goat anti-rabbitIgG-HRP antibody (1:25000) and the ECL system.

-   (B) In each case 40 μg of total protein from the C terminus (1) and    N terminus (2) of HC110-R induced in E. coli, and untransfected    HEK-293 cells (3) and HEK-293 cells stably transfected with    HC110-R-Myc/His (4) and adult H. contortus nematodes (5) were    fractionated in a 10% SDS polyacrylamide gel and partially renatured    with a 4 M urea-containing renaturation buffer. The blot was blocked    overnight, incubated with 20 nM pure α-LTX, and α-LTX-binding    proteins were detected with a rabbit anti-α-LTX IgG antibody    (1:5000), biotin-protein A (1:100), streptavidin-peroxidase (1:3000)    and the ECL system.

FIG. 10: Dose-dependence of α-LTX on the HC110-R-mediated Ca²⁺signalling

48 h after transient transfection with HC110-R-GFP, HEK-293 cells wereloaded with 1 μM Fura-2/AM for 30 min. The cells were stimulated 6 minafter the start of the measurement with various α-LTX concentrations fora further 44 min. The 340/380 nm quotient (ratio) was measured as afunction of the time. The number of selected cells is indicated by n.

-   (A) HC110-R-GFP-expressing cells were stimulated with 7.5 nM (black    line) or 25 nM α-LTX (grey line).-   (B) 50 nM (grey line) or 75 nM α-LTX (black line) was added to    HC110-R-GFP-expressing cells.-   (C)HC110-R-GFP-expressing cells were stimulated with 90 nM (grey    line) or 120 nM α-LTX (black line).-   (D) [Ca²⁺]_(i) is plotted in nM on the abscissa, and the α-LTX    concentrations (0, 7.5, 25, 50, 75, 90 and 120 nM) for the first    rapid peak (1st peak, grey line) and the second delayed peak (2nd    peak, black line) employed to stimulate HC110-R-GFP-expressing    HEK-293 cells are plotted on the ordinate.

FIG. 11: Ca²⁺ Imaging of HC110-R-transfected HEK 293 cells

-   (A) HC110-R with C-terminal GFP tag (HC110-R-GFP): black line.    -   HC110-R With N-terminal GFP tag (GFP-HC110-R): grey line.-   (B) 75 nM α-LTX with untransfected HEK-293 cells (n. t., black line)    and GFP-transfected cells (GFP, grey line).-   (C) 75 nM α-LTX with cells transfected with C-terminally tagged    human M1 muscarinic acetylcholine receptor (M1-R-GFP, grey line) and    with C-terminally tagged mouse β₂-adrenergic acetylcholine receptor    (β₂-R-GFP, black line).-   (D) HC110-R-GFP-transfected cells were treated with 2 mM EGTA 6    minutes before addition of α-LTX (75 nM) (+EGTA, grey line). EGTA    was omitted from the control (−EGTA, black line).-   (E) 2 mM EGTA were added 10 min after addition of α-LTX (75 nM)    (+EGTA, grey line). No EGTA was added to the control mixture (−EGTA,    black line).-   (F) As controls, untransfected (n. t., grey line) and    HC110-R-GFP-expressing cells (black line) were mixed with 2 nM EGTA    for 30 minutes.

FIG. 12: Interference by BAY44-4400 with signal transmission caused byα-LTX

HEK-293 cells were transiently transfected with GFP-tagged HC110-Rprotein and stimulated (arrows) 48 h after the transfection. Ca²⁺imaging was carried out for 50 minutes (Figs. A-D), control experimentswith various endogenous natural receptors of untransfected HEK-293 cellsfor 20 minutes. n=number of cells.

-   (A) Addition of 400 ng/ml BAY44-4400 (black line) or of PF1022-001    (grey line) to the cells. As controls, the cells were treated with    Na⁺ HBS/HEPES buffer (HBS), which also served as solvent for the    respective substances, after the adddition of BAY44-4400 or    PF1022-001.-   (B) Signal transmission caused by α-LTX (75 nM) in the presence of 4    ng/ml BAY44-4400 (black line) and PF1022-001 (grey line).-   (C) Signal transmission caused by α-LTX in cells preincubated with 4    ng/ml BAY44-4400 (black line) or PF1022-001 (grey line) for 90    minutes. The substances were removed before addition of α-LTX.-   (D) Signal transmission caused by α-LTX in HC110-R-GFP-transfected    cells preincubated with 400 ng/ml BAY44-4400 (black line) or    PF1022-001 (grey line) for 90 minutes. The substances were removed    before addition of α-LTX.-   (E) Untransfected HEK-293 cells were stimulated with 1 mM    carbamylcholine chloride (carbachol) for 100 s in the presence (400    ng/ml BAY44-4400, black line) or absence of 400 ng/ml BAY44-4400    (grey line, -BAY44-4400).-   (F) Untransfected HEK-293 cells were stimulated with 1 mM    isoproterenol and with 1 mM arecoline in each case for 100 s in the    presence (400 ng/ml BAY 44-4400, black line) or absence of 400 ng/ml    BAY44-4400 (-BAY44-4400, grey line).

FIG. 13: Detection of PF1022A binding to HC110-R-transfected HEK-293cells by FACScan

In each case 5×10⁵ HEK-293 cells which were not transfected, weretransiently transfected with GFP, HC110-R-GFP, GFP-HC110-R, β₂-R-GFP andM1-R-GFP, and transiently cotransfected with HC110-R-Myc/His and GFPwere incubated, 24 h after the transfection, with 0.5 μg/mlPF1022A-biotin and streptavidin-phycoerythrin (1:300) and then fixed. Asnegative controls, HC110-R-GFP-transfected cells were incubated onlywith streptavidin-phycoerythrin, without PF1022A-biotin. Aftersubtraction of the autofluorescence, i.e. of the 4.6% untransfectedcells (n. t.) in the green channel from all green-fluorescent cells andof the 4.4% nonspecific staining caused by streptavidin-phycoerythrin inthe red channel, the percentage of GFP-fluorescent cells (GFP,HC110-R-GFP, GFP-HC110-R, β₂-R-GFP, M1-R-GFP and HC110-R-Myc/His+GFP)which have bound PF1022A was found (PF1022A binding in %). The standarddeviation is derived by determining the means of triplicate mixtures.

FIG. 14: Effect of BAY44-4400 and the optical antipode PF1022-001 on theHC110-R-mediated Ca²⁺ signalling

Loading of the cells with 1 μM Fura-2/AM took place in untransfectedHEK-293 cells and 48 h after transient transfection with HC110-R-GFP orM1-R-GFP. The quotient of 340/380 nm (ratio) was measured as a functionof the time. The number of selected cells is indicated by n.

-   (A) Untransfected (n. t., grey line) and HC110-R-GFP-transfected    (HC110-R-GFP, black line) cells were mixed with 0.1% of the solvent    DMSO in Na⁺ HBS buffer after 6 min as control.-   (B) HC110-R-GFP- (black line) and M1-R-GFP- (grey line) transfected    cells were stimulated with 400 ng/ml BAY44-4400 after 6 min.-   (C) As (B), HC110-R-GFP- (black line) and M1-R-GFP- (grey line)    transfected cells were stimulated with 400 ng/ml PF1022-001 after 6    min.-   (D) HC110-R-GFP-expressing cells were preincubated with 400 ng/ml    BAY44-4400 (black line) or PF1022-001 (grey line) for 90 min, and    unbound substances were washed out before the measurement.-   (E) 10 μM piperazine were added to untransfected (n. t., grey line)    and HC110-R-GFP-expressing cells (HC110-R-GFP, black line) after 6    min.-   (F) As (E), but a mixture of 10 μM piperazine and 400 ng/ml    BAY44-4400 was administered to untransfected (n. t., grey line) and    HC110-R-GFP-expressing cells (HC110-R-GFP, black line) after 6 min.

FIG. 15: Interactions of the HC110-R-mediated α-LTX signalling throughPF1022A derivatives

Loading of the cells with 1 μM Fura-2/AM took place in untransfectedHEK-293 cells and 48 h after transient transfection with HC110-R-GFP orM1-R-GFP. The amount of α-LTX employed was always 75 nM. For theexperiments with the control substances, the cells were flushed at aflow rate of 1.6 ml Na⁺ HBS/min. The quotient of 340/380 nm (ratio) wasmeasured as a function of the time. The number of selected cells isindicated by n.

-   (A) HC110-R-GFP- (black line) and M1-R-GFP- (grey line) expressing    cells were mixed with 0.1% DMSO 6 min before α-LTX stimulation in    order to preclude DMSO having an effect on α-LTX-induced changes in    [Ca²⁺]_(i).-   (B) HC110-R-GFP-transfected cells were mixed with 4 ng/ml BAY44-4400    (black line) or PF1022-001 (grey line) 6 min before addition of    α-LTX.-   (C) As (B), but 400 ng/ml BAY44-4400 (black line) or PF1022-001    (grey line) were added to HC110-R-GFP-expressing cells 6 min before    stimulation with α-LTX.-   (D) HC110-R-GFP-transfected cells were preincubated with 4 ng/ml    BAY44-4400 (black line) or PF1022-001 (grey line) for 90 min, and    unbound substances were removed before the measurement. Stimulation    of the cells took place after 6 min with α-LTX.-   (E) As (D), but HC110-R-GFP-transfected cells were preincubated with    400 ng/ml BAY44-4400 (black line) or PF1022-001 (grey line) for 90    min, unbound substances were removed before the measurement, and the    cells were stimulated with α-LTX after 6 min.-   (F) Cells stably expressing HC110-R-Myc/His were mixed with 400    ng/ml BAY44-4400 (black line) or PF1022-001 (grey line) 6 min before    addition of α-LTX.-   (G) As controls, 1 mM carbamylcholine chloride (carbachol) was added    after 6 min for 100 s, and washed out again with Na⁺ HBS, to    untransfected cells in the presence (400 ng/ml BAY44-4400, black    line) or absence (-BAY44-4400, grey line) of 400 ng/ml BAY44-4400.-   (H) As (G), but M1-R-GFP-transfected cells were stimulated in the    presence (400 ng/ml BAY44-4400, black line) or absence (-BAY44-4400,    grey line) of 400 ng/ml BAY44-4400 after 6 min with 0.1 mM carbachol    for 100 s and washed out again with Na⁺ HBS.-   (I) As controls, untransfected cells were stimulated in the presence    (400 ng/ml BAY44-4400, black line) or absence (-BAY44-4400, grey    line) of 400 ng/ml BAY44-4400 after 3 min for 100 s with 1 mM    isoproterenol and after 12 min for 100 s with 1 mM arecoline. The    substances were washed out again with Na⁺ HBS after the 100 s.

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1. Use of calcium channel transmembrane receptors from helminths foridentifying substances with anthelmintic activity.
 2. Use of calciumchannel transmembrane receptors from arthropods for identifyingsubstances with arthropodicidal activity.
 3. Use of transmembranereceptors according to claim 1 or 2, characterized in that they areG-protein-coupled transmembrane receptors with seven transmembranedomains.
 4. Use of transmembrane receptors according to any of claims 1to 3, characterized in that they are transmembrane receptors of thesecretin subfamily.
 5. Use of transmembrane receptors according to anyof claims 1, 3 or 4, characterized in that they are transmembranereceptors from nematodes.
 6. Use of transmembrane receptors according toany of claims 2 to 4, characterized in that they are transmembranereceptors from acarina.
 7. Use of transmembrane receptors according toany of claims 1 and 3 to 5, characterized in that the transmembranereceptor HC110-R from Haemonchus contortus is involved.
 8. Use oftransmembrane receptors according to any of claims 1 to 6, characterizedin that they are homologous to the transmembrane receptor HC110-R. 9.Use of transmembrane receptors according to any of claims 1 to 8 for Identifying calcium channel blockers.
 10. Use of transmembrane receptorsaccording to any of claims 1 to 8 for identifying calcium channelblockers, characterized in that they are alpha-latrotoxin-bindingtransmembrane receptors.
 11. Use of alpha-latrotoxin as agonist oftransmembrane receptors according to any of claims 1, 3 to 5 and
 7. 12.Use of alpha-latrotoxin as nematicide.
 13. Use of alpha-latrotoxin inmethods for identifying compounds having nematicidal and/orarthropodicidal activity.
 14. Method for obtaining the HC110-R receptorand proteins homologous thereto, comprising the expression of thepolypeptide or fragments thereof in a prokaryotic or eukaryoticexpression system.
 15. Method according to claim 14, characterized inthat a eukaryotic expression system is involved.
 16. Method according toclaim 15, characterized in that HEK 293 or COS7 cells are used for theexpression.
 17. Host cells which make transient expression of thereceptors HC110-R and proteins homologous thereto possible.
 18. Hostcells which make stable expression of the receptors HC110-R and proteinshomologous thereto possible.
 19. Host cells according to claim 18,characterized in that they express aequorin.
 20. Host cells according toclaim 18, characterized in that they are of the stably transformed cellline with the deposit number DSM ACC2464.
 21. Host cells according toclaim 18 or 19, characterized in that they are of the stably transformedcell line with the deposit number DSM ACC2465.
 22. Vectors for stabletransformation of host cells according to any of claims 18 to 21 and fortransient transformation of host cells according to claim
 17. 23.Vectors for stable transformation of host cells according to claim 18 or20, characterized in that the vector pMyc6×His is involved.
 24. Methodfor identifying agonists and/or antagonists of calcium channeltransmembrane receptors from helminths and/or arthropods, comprising thefollowing steps: a) bringing a host cell according to any of claims 17to 21 or membranes thereof into contact with a chemical compound or amixture of chemical compounds under conditions which permit interactionof a chemical compound with the polypeptide, and b) determining thechemical compound which specifically binds to the polypeptide. 25.Method for finding compounds which alter the expression of calciumchannel transmembrane receptors from helminths or arthropods, comprisingthe following steps: a) bringing a host cell according to any of claims17 to 21 into contact with a chemical compound or a mixture of chemicalcompounds, b) determining the calcium channel transmembrane receptorconcentration, and c) determining the compound which specificallyaffects the expression of the polypeptide.
 26. Method according to claim24 or 25, characterized in that G-protein-coupled transmembranereceptors of the secretin subfamily or fragments thereof are involved.27. Method according to claim 24 or 25, characterized in that thetransmembrane receptor HC110-R or fragments thereof and proteinshomologous thereto are involved.
 28. Method according to any of claims24, 26 and 27, characterized in that the test substance is brought intocontact with the transmembrane receptor under conditions which permitinteraction of the receptor molecules with the test substance, and thena) binding of the test substance which has taken place is detected, andb) the activity of the receptor molecule in the presence of the testsubstance and its activity in the absence of a test substance arecompared.
 29. Method according to any of claims 24 to 28, characterizedin that a cell-based test system is used.
 30. Method according to claim29, characterized in that cells according to any of claims 17 to 21 areused.
 31. Method according to any of claims 24 and 26 to 28,characterized in that a cell-free test system is used.
 32. Methodaccording to any of claims 24 and 26 to 31, characterized in that theinteraction of a test substance with the transmembrane receptor isdetected through the displacement of alpha-latrotoxin bound thereto. 33.Method according to any of claims 24 and 26 to 31, characterized in thatthe interaction of a test substance with the transmembrane receptor isdetected through the displacement of nifedipine bound thereto.
 34. Useof nifedipine in a method according to any of claims 24 and 26 to 31.35. Substances identified in a method according to any of claims 24 to34.
 36. Use of substances according to claim 35 for producing acomposition for controlling helminths and/or arthropods.
 37. Use ofmodulators of the HC110-R receptor and proteins homologous thereto asanthelmintics and/or arthropodicides.
 38. Use of DNA coding for theHC110-R receptor and DNA homologous thereto for producing transgenicinvertebrates.
 39. Transgenic invertebrates which comprise the HC110-Rreceptor or proteins homologous thereto.
 40. Transgenic invertebratesaccording to claim 39, characterized in that they are Drosophilamelanogaster and Caenorhabditis elegans.
 41. Use of DNA oligonucleotideswhich specifically hybridize onto the DNA coding for the HC110-Rreceptor for detecting DNA derived from helminths.
 42. Method fordetecting DNA from helminths, characterized in that a) DNAoligonucleotides which hybridize onto the DNA coding for the HC110-Rreceptor or strands complementary thereto or onto the 5′- or 3′-flankingregions thereof are made available, b) the DNA oligonucleotides arebrought into contact with a DNA-containing sample, c) the hybridizationof the DNA oligonucleotide is detected, d) the detected sequence issequenced, and e) the sequence is compared with the DNA sequence codingfor the HC110-R receptor.
 43. Diagnostic test kit comprising a DNAsequence coding for the HC110-R receptor, or fragment thereof or DNAsequences homologous thereto.
 44. Diagnostic test kit according to claim43, characterized in that the DNA sequences are provided with adetectable marker.
 45. Use of the HC110-R receptor or fragments thereofand proteins homologous thereto for producing vaccines.